Immunosuppressive agents and their use in therapy

ABSTRACT

In certain aspects the agent and compositions of the invention may be used as single agents. In other aspects of the invention agents and compositions of the invention may be used in conjunction with one or more additional active agents, such as kinase inhibitors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Phase application filed under 35 U.S.C § 371 of PCT International Application No. PCT/EP2014/073966 with an International Filing Date of Nov. 6, 2014, which claims under 35 USC 119(a) the benefit of United Kingdom Application No. 1319620.9, filed Nov. 6, 2013. The entire contents of the PCT International Application No. PCT/EP2014/073966 are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 7, 2019, is named 050955-501N01US_SL.txt and is 268 KB in size.

The present invention relates to novel agents, particularly peptides or mimetics thereof and their encoding nucleic acids, pharmaceutical compositions comprising at least one of said agents, and their use in the suppression of cellular immune system responses and/or the treatment of infectious diseases. The agents find particular use in the treatment of disorders or conditions associated with cytokine release from non-proliferating immune cells, e.g. unwanted, undesirable or excessive cytokine release. The agents may be useful alone or in combination with other therapeutically active compounds, such as immunosuppressive compounds, anti-inflammatory compounds, anti-microbial compounds etc. Such compounds may be, for instance, antagonists or inhibitors of signal transduction pathways, particularly toll-like receptor signalling pathways, such as toll-receptor antagonists and/or protein kinase inhibitors. Also provided are therapeutic methods which comprise the use of said agents for the aforementioned uses. The agents may also be used in the manufacture or preparation of medicaments for the aforementioned therapies.

APIM peptides are a group of peptides that interact with PCNA (proliferating cell nuclear antigen) via a novel PCNA interacting motif (Gilljam et al., 2009. Identification of a novel, widespread, and functionally important PCNA-binding motif, J. Cell Biol. 186(5), pp. 645-654). The motif has been termed APIM (AlkB homologue 2 (hABH2) PCNA-interacting motif) since it was first identified as mediating the interaction between hABH2 and PCNA, but as will be clear from the disclosure below, APIM motifs have now been identified in a wide range of proteins. The, PCNA binding motif found in APIM peptides typically is defined using the consensus sequence, [R/K]-[F/W/Y]-[L/I/V/A]-[L/I/V/A]-[K/R] (SEQ ID NO:19).

PCNA is a member of the sliding clamp family of proteins, which is known to be involved in both DNA replication and DNA repair. The main function of PCNA is to provide replicative polymerases with the high processivity needed for duplication of the genome. In live S-phase cells, PCNA tagged with green fluorescent protein (GFP) forms distinct foci representing sites of replication. It can therefore be used as an S-phase marker.

Numerous proteins involved in cellular processes such as DNA repair, chromatin assembly, epigenetic and chromatin remodelling, sister-chromatid cohesion, cell cycle control and survival are localised in so-called replication factories which contain more than a dozen replication forks. Many of these proteins interact with PCNA and have been shown to co-localise with PCNA in replication foci. PCNA-interacting proteins have particularly been thought to be involved in DNA repair and replication and cell proliferation.

Thus, various proteins interact with PCNA and it is thought that many of these interactions are mediated via a conserved PCNA interacting peptide sequence called the PIP-box (QxxL/I/MxxF/DF/Y [SEQ ID NO: 1205]), wherein x can be any amino acid. However, peptides that contain a PIP-box typically are extremely cytotoxic and therefore unsuitable for use in therapy.

However, APIM peptides have been shown to be useful in therapy. Specifically APIM peptides have been shown to be effective in sensitizing cells to cytotoxic and cytostatic agents, particularly DNA-damaging agents (WO 2009/104001) and indeed as an apoptosis-inducing cytotoxic agent in its own right (Müller et al., 2013. Targeting Proliferating Cell Nuclear Antigen and Its Protein Interactions Induces Apoptosis in Multiple Myeloma Cells, PLOS One, 8(7), e70430, pp. 1-12). Thus, APIM peptides have been shown to be useful in combination with cytotoxic and/or cytostatic agents in the treatment of a disorder or condition where it is desirable to inhibit the growth of cells, or in a treatment which involves cytostatic therapy, i.e. to prevent or inhibit the unwanted proliferation of cells.

In work leading up to the present invention, it was determined that PCNA is present in the cytosol in some cell types, particularly terminally differentiated cells, where it is thought it interacts with a variety of cytosolic polypeptides in the regulation of apoptosis, e.g. via caspases. Accordingly, PCNA was not expected to be present in the cytosol of non-proliferating cells, particularly cells that are not terminally differentiated.

However, the inventors have surprisingly found that APIM peptides have an effect on non-proliferating cells. Additionally, APIM peptides have been shown by the inventors to interfere with protein interactions between proteins involved in various signal transduction pathways, particularly toll-like receptor signal transduction pathways. As shown in more detail in the Examples, the inventors determined that APIM peptides can reduce the release of pro-inflammatory cytokines from non-proliferating immune cells, e.g. non-terminally differentiated immune cells, such as monocytes, without inducing apoptosis.

Toll-like receptors (TLRs) form one of the major classes of transmembrane pattern-recognition receptors (PRRs). In this respect, cells of the innate immune system recognize pathogen associated molecular patterns (PAMPs) through PRRs. In humans, ten different TLRs have been identified that recognize a variety of PAMPs.

TLR1, 2, 4, 5, and 6 are localized at the cell surface and detect mainly bacterial cell wall components, such as lipopolysaccharide (LPS) from gram-negative bacteria. LPS is recognized by TLR4. TLR3, 7, 8, and 9 localize to intracellular membrane-bound compartments, including endolysosomes, where they recognize viral and bacterial nucleic acids. TLR3 recognizes double-stranded RNA (dsRNA) from viruses and the synthetic dsRNA analog polyinosinic-polycytidylic acid (polyIC).

Once activated, PRRs induce intracellular signal transduction pathways that result in the expression and secretion of cytokines and chemokines, which function to coordinate the immune response in-host defence against microbial pathogens.

Upon ligand binding, TLRs recruit the main adaptor proteins MyD88 and/or TRIF and activate signal transduction pathways that trigger the expression and secretion of cytokines needed to induce an immune response. The MyD88-dependent pathway is used by all TLRs except TLR3. Activation of the MyD88 pathway induces the production of inflammatory cytokines via the transcription factor NF-κB and mitogen-activated protein kinases (MAPKs). TLR3 and TLR4 both recruit the adaptor TRIF and induce type I interferons (IFNs) via the transcription factor IRF3. In addition, TRIF activates NF-κB and MAPKs.

TLR signalling also activates the phosphatidylinositide 3-kinase (PI3K)/Akt pathway. It has been suggested that PI3K/Akt activity is required for full activation of IRF3 in the TRIF-dependent pathway during TLR3 and 4 signalling. In addition to TLR3, cytosolic receptors including RIG-1, MDA5 and PKR also recognize dsRNA and mediate antiviral responses via signaling pathways similar to TLRs.

Thus, various signalling pathways are involved in coordinating the responses of the innate cellular immune defence system, which is central to surviving microbial and parasitic challenges. This system is also relevant for managing deviant cells after viral infections and in autoimmune diseases. The system uses cytokines and chemokines to communicate and coordinate immune responses and these signals are immensely important in controlling local and systemic responses. However, cytokines and chemokines have been linked to a variety of diseases and disorders, typically as a result of abnormal levels of these molecules, which can trigger undesirable responses in vivo. For instance, the over-secretion of cytokines, such as in a so-called “cytokine storm”, can have severe adverse effects on health.

Cytokines and chemokines are peptide, protein or glycoprotein signalling molecules that are used extensively in cellular communication. The terms cytokine and chemokine are used interchangeably herein and encompass large and diverse families of molecules that are produced widely throughout the body. Whilst virtually all nucleated cells are capable of producing cytokines and chemokines, immune cells are a particularly important source of cytokines and chemokines. Immune cells may be identified and characterised, at least to some extent, by the mixture of cytokines and chemokines that they secrete, as this forms a crucial part of their role in the immune system.

Monocytes are a type of immune cell (leukocyte or white blood cell) that are produced by the bone marrow from hematopoietic stem cell precursors called monoblasts. Monocytes are non-proliferating cells that are not terminally differentiated. Accordingly, monocytes are primarily found in blood and the spleen, and typically constitute between about 3-8% of the leukocytes in the blood. These cells tend to circulate in the bloodstream for about one to three days before moving into tissues throughout the body, where they terminally differentiate into different types of macrophages, depending on the anatomical location.

Monocytes characteristically display the cell surface receptor CD14 and may also express and display the cell surface receptor CD16. These cells are particularly associated with the release of pro-inflammatory cytokines and chemokines such as TNFα, IL1RA, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, CCL11, BasicFGF, G-CSF, GM-CSF, INFγ, CXCL10, CCL2, CCL3, CCL4, PDGF-β, CCL5 and VEGF, particularly following stimulation by components of microbial cells (such as LPS) and/or viruses (such as double stranded nucleic acids). Monocytes are often found at very high levels in subjects with severe infections, e.g. in cases of sepsis and septicaemia, where it is thought that overproduction of cytokines and chemokines contributes to symptoms and complications associated with these conditions, e.g. multiple organ dysfunction syndrome (i.e. multiple organ failure).

Indeed uncontrolled or excessive cytokine and/or chemokine release (e.g. hypercytokinemia) from immune cells in blood, such as monocytes, is associated with a variety of disorders. For instance, it is thought that the excess release of pro-inflammatory cytokines and/or chemokines is the primary driving force of disease and death caused by Plasmodium falciparum infections, i.e. malaria. Other infectious diseases, including bacterial, fungal and some viral infections (e.g. influenza, such as swine influenza, avian influenza), may result in excessive pro-inflammatory cytokine and/or chemokine release in blood. Such infections may be local or systemic. In addition, allogenic tissue transplants can result in graft versus host disease, in which immune cells produce excess levels of cytokines and chemokines, such as TNFα.

Therapies for disorders caused by, or associated with, excessive release of cytokines and chemokines have typically focussed on the inhibition of particular cytokines and chemokines or small groups of related cytokines and chemokines. For instance, therapies may utilize specific binding proteins, such as antibodies or soluble receptors, to neutralise the released and undesirable cytokines and chemokines. Alternatively, binding proteins or small molecule antagonists may be used to block cytokine and chemokine receptors to prevent downstream signalling. However, a combination of such therapies may be required to deal with an excess of several cytokines and chemokines, and combining multiple therapeutic agents can be expensive and may have undesirable and unpredictable side effects. Whilst the use of corticosteroids has been suggested for use in subjects suffering from hypercytokinemia, there is limited evidence that such treatments are effective. Furthermore, therapies to date have centred on the treatment of particular tissues, such as the lungs, e.g. the treatment of COPD (chronic obstructive pulmonary disease) by, for instance, inhibiting G-CSF (Granulocyte colony-stimulating factor).

Accordingly, there remains a need for effective therapies suitable for the treatment of diseases, disorders or conditions caused by, or associated with, excessive release of cytokines and chemokines (e.g. infectious diseases), which also have minimal side effects.

As mentioned above, the inventors have surprisingly found that APIM peptides can reduce the release of pro-inflammatory cytokines and chemokines from non-proliferating immune cells, e.g. non-terminally differentiated immune cells, such as monocytes, without inducing apoptosis. Thus, the inventors have determined that APIM peptides may function via a general mechanism on non-proliferating immune cells to inhibit (e.g. reduce, diminish, lessen etc) the release of cytokines and/or chemokines from said cells, e.g. when the cells have been stimulated with any one of a variety of components, e.g. components from infectious entities (microbial, viral and parasitic), such as LPS or double stranded nucleic acid.

These surprising findings have led the inventors to propose new therapeutic uses for APIM peptides, i.e. peptides comprising a PCNA binding motif, namely for use in treating a condition or disorder associated with cytokine release from non-proliferating immune cells in blood, particularly conditions or disorders associated with excessive, undesirable or uncontrolled pro-inflammatory cytokine release from non-proliferating immune cells in blood, e.g. an infectious disease or infection or a disease or condition exacerbated or caused by an infection.

Alternatively viewed, the inventors propose the use of APIM peptides for treating or preventing excessive, undesirable or uncontrolled pro-inflammatory cytokine release from non-proliferating immune cells in blood in a subject, e.g. a subject with an infectious disease or infection or a disease or condition exacerbated or caused by an infection.

Thus, at its broadest, the invention can be seen to provide a method of treating or preventing a disorder or condition associated with cytokine release from non-proliferating immune cells in blood, particularly a disorder or condition which results in or from, or is associated with, hypercytokinemia, said method comprising administering (particularly administering an effective amount of) an agent comprising or encoding a peptide comprising an APIM motif or a composition (e.g. a pharmaceutical composition) containing an agent comprising or encoding a peptide comprising an APIM motif to a subject in need thereof.

Thus, the invention provides an agent comprising or encoding a peptide comprising an APIM motif or a composition (e.g. a pharmaceutical composition) containing an agent comprising or encoding a peptide comprising an APIM motif, for use in treating or preventing a disorder or condition associated with cytokine release from non-proliferating immune cells in blood, particularly a disorder or condition resulting in or from, or associated with, hypercytokinemia.

In another aspect, there is provided the use of an agent comprising or encoding a peptide comprising an APIM motif in the manufacture of a medicament for the treatment or prevention of a disorder or condition associated with cytokine release from non-proliferating immune cells in blood, particularly a disorder or condition resulting in or from, or associated with, hypercytokinemia.

More particularly, the invention provides a method of treating or preventing a disorder or condition associated with cytokine release from non-proliferating immune cells in blood, particularly a disorder or condition which results in or from, or is associated with, hypercytokinemia, said method comprising administering (particularly administering an effective amount of) an agent or a composition (e.g. a pharmaceutical composition) containing an agent to a subject in need thereof, wherein said agent comprises:

(i) an oligopeptidic compound comprising a PCNA interacting motif and a domain that facilitates the cellular uptake of said compound, wherein the PCNA interacting motif is X₁X₂X₃X₄X₅ (SEQ ID NO: 1) and wherein:

X₁ is a basic amino acid;

X₂ is an aromatic amino acid;

X₃ is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P);

X₄ is any amino acid other than Proline (P), an acidic amino acid or an aromatic amino acid; and

X₅ is a basic amino acid or Proline (P); or

(ii) a nucleic acid molecule comprising a sequence encoding the oligopeptidic compound of (i).

In another aspect, there is provided an agent or a composition (e.g. a pharmaceutical composition) containing an agent for use in treating or preventing a disorder or condition associated with cytokine release from non-proliferating immune cells in blood, particularly a disorder or condition resulting in or from, or associated with, hypercytokinemia, wherein said agent comprises:

(i) an oligopeptidic compound comprising a PCNA interacting motif and a domain that facilitates the cellular uptake of said compound, wherein the PCNA interacting motif is X₁X₂X₃X₄X₅ (SEQ ID NO: 1) and wherein:

X₁ is a basic amino acid;

X₂ is an aromatic amino acid;

X₃ is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P);

X₄ is any amino acid other than Proline (P), an acidic amino acid or an aromatic amino acid; and

X₅ is a basic amino acid or Proline (P); or

(ii) a nucleic acid molecule comprising a sequence encoding the oligopeptidic compound of (i).

In a further aspect, there is provided the use of an agent in the manufacture of a medicament for the treatment or prevention of a disorder or condition associated with cytokine release from non-proliferating immune cells in blood, particularly a disorder or condition resulting in or from, or associated with, hypercytokinemia, wherein said agent comprises:

(i) an oligopeptidic compound comprising a PCNA interacting motif and a domain that facilitates the cellular uptake of said compound, wherein the PCNA interacting motif is X₁X₂X₃X₄X₅ (SEQ ID NO: 1) and wherein:

X₁ is a basic amino acid;

X₂ is an aromatic amino acid;

X₃ is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P);

X₄ is any amino acid other than Proline (P), an acidic amino acid or an aromatic amino acid; and

X₅ is a basic amino acid or Proline (P); or

(ii) a nucleic acid molecule comprising a sequence encoding the oligopeptidic compound of (i).

In particular embodiments the invention provides a method of treating or preventing hypercytokinemia resulting from cytokine release from non-proliferating immune cells in blood, said method comprising administering an agent, or a composition containing an agent, to a subject in need thereof, wherein said agent comprises:

(i) an oligopeptidic compound comprising a PCNA interacting motif and a domain that facilitates the cellular uptake of said compound, wherein the PCNA interacting motif is X₁X₂X₃X₄X₅ (SEQ ID NO: 1) and wherein:

X₁ is a basic amino acid;

X₂ is an aromatic amino acid;

X₃ is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P);

X₄ is any amino acid other than Proline (P), an acidic amino acid or an aromatic amino acid; and

X₅ is a basic amino acid or Proline (P); or

(ii) a nucleic acid molecule comprising a sequence encoding the oligopeptidic compound of (i).

In a further embodiment, the invention provides an agent, or composition containing an agent, for use in treating or preventing hypercytokinemia in a subject resulting from cytokine release from non-proliferating immune cells in blood, wherein said agent comprises:

(i) an oligopeptidic compound comprising a PCNA interacting motif and a domain that facilitates the cellular uptake of said compound, wherein the PCNA interacting motif is X₁X₂X₃X₄X₅ (SEQ ID NO: 1) and wherein:

X₁ is a basic amino acid;

X₂ is an aromatic amino acid;

X₃ is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P);

X₄ is any amino acid other than Proline (P), an acidic amino acid or an aromatic amino acid; and

X₅ is a basic amino acid or Proline (P); or

(ii) a nucleic acid molecule comprising a sequence encoding the oligopeptidic compound of (i).

In still further embodiments, the invention provides the use of an agent in the manufacture of a medicament for the treatment or prevention of hypercytokinemia in a subject resulting from cytokine release from non-proliferating immune cells in blood, wherein said agent comprises:

(i) an oligopeptidic compound comprising a PCNA interacting motif and a domain that facilitates the cellular uptake of said compound, wherein the PCNA interacting motif is X₁X₂X₃X₄X₅ (SEQ ID NO: 1) and wherein:

X₁ is a basic amino acid;

X₂ is an aromatic amino acid;

X₃ is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P);

X₄ is any amino acid other than Proline (P), an acidic amino acid or an aromatic amino acid; and

X₅ is a basic amino acid or Proline (P); or

(ii) a nucleic acid molecule comprising a sequence encoding the oligopeptidic compound of (i).

In some embodiments, the agent as defined herein may be used in combination with one or more additional active agents, e.g. other therapeutically active agents, such as immunosuppressive compounds, anti-inflammatory compounds, anti-microbial compounds, steroids (e.g. corticosteroid), kinase inhibitors (such as p38 MAPK inhibitors, class I PI3K inhibitors) etc., in order to enhance the effect of the agent. However, in some embodiments, the agent as defined herein may be used alone, i.e. as the only active agent in a composition and/or medicament.

Accordingly, in yet another aspect, there is provided a method of treating or preventing a disorder or condition associated with cytokine release from non-proliferating immune cells in blood, particularly a disorder or condition which results in or from, or is associated with, hypercytokinemia, said method comprising administering (particularly administering an effective amount of) an agent or a composition (e.g. a pharmaceutical composition) containing an agent and separately, simultaneously or sequentially administering of one or more additional active agents, e.g. a therapeutically active agent, to a subject in need thereof, wherein said agent comprises:

(i) an oligopeptidic compound comprising a PCNA interacting motif and a domain that facilitates the cellular uptake of said compound, wherein the PCNA interacting motif is X₁X₂X₃X₄X₅ (SEQ ID NO: 1) and wherein:

X₁ is a basic amino acid;

X₂ is an aromatic amino acid;

X₃ is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P);

X₄ is any amino acid other than Proline (P), an acidic amino acid or an aromatic amino acid; and

X₅ is a basic amino acid or Proline (P); or

(ii) a nucleic acid molecule comprising a sequence encoding the oligopeptidic compound of (i).

In a particular embodiment, the invention provides a method of treating or preventing hypercytokinemia resulting from cytokine release from non-proliferating immune cells in blood, said method comprising administering (particularly administering an effective amount of) an agent as defined herein or a composition (e.g. a pharmaceutical composition) containing an agent as defined herein and separately, simultaneously or sequentially administering of one or more additional active agents, e.g. a therapeutically active agent, to a subject in need thereof.

Alternatively viewed, there is provided an agent or composition (e.g. a pharmaceutical composition) as defined herein for use in combination with one or more additional active agents, e.g. a therapeutically active agent, in the treatment or prevention of a disorder or condition associated with cytokine release from non-proliferating immune cells in blood, particularly a disorder or condition which results in or from, or is associated with, hypercytokinemia.

For instance, there is provided an agent or composition (e.g. a pharmaceutical composition) as defined herein for use in combination with one or more additional active agents, e.g. a therapeutically active agent, in the treatment or prevention of hypercytokinemia in a subject resulting from cytokine release from non-proliferating immune cells in blood.

Thus, also provided is the use of an agent as defined herein in the manufacture of a medicament for use in combination with one or more additional active agents, e.g. a therapeutically active agent, in the treatment or prevention of a disorder or condition associated with cytokine release from non-proliferating immune cells in blood, particularly a disorder or condition which results in or from, or is associated with, hypercytokinemia.

For instance, in some embodiments the invention provides the use of an agent as defined herein in the manufacture of a medicament for use in combination with one or more additional active agents, e.g. a therapeutically active agent, in the treatment or prevention of hypercytokinemia in a subject resulting from cytokine release from non-proliferating immune cells in blood.

Thus, in one embodiment the medicament may further comprise one or more additional active agents, such as a therapeutically active agent that is useful in treating or preventing the disease or condition (or the symptoms of, or associated with the disease or condition, e.g. hypercytokinemia), associated with cytokine release from non-proliferating immune cells in blood. The therapeutically active agent may be, but is not limited to, an immunosuppressive compound, an anti-inflammatory compound, anti-microbial compound, a steroid (e.g. a corticosteroid) or a kinase inhibitor (such as a p38 MAPK inhibitor or a class I PI3K inhibitor).

The medicament may be in the form of a single composition (e.g. a pharmaceutical composition) comprising both the agent as defined herein and the one or more additional active agents, e.g. therapeutically active agent, or it may be in the form of a kit or product containing them for separate (e.g. simultaneous or sequential) administration.

There is thus also provided the use of an agent as defined herein in the manufacture of a medicament for the treatment or prevention of a disorder or condition associated with cytokine release from non-proliferating immune cells in blood, particularly a disorder or condition which results in or from, or is associated with, hypercytokinemia, wherein the medicament is administered separately, simultaneously or sequentially with one or more additional active agents, e.g. a therapeutically active agent.

Thus, in some embodiments, there is provided the use of an agent as defined herein in the manufacture of a medicament for the treatment or prevention hypercytokinemia in a subject resulting from cytokine release from non-proliferating immune cells in blood, wherein the medicament is administered separately, simultaneously or sequentially with one or more additional active agents, e.g. a therapeutically active agent.

In another aspect, the invention provides a product or kit containing an agent as defined herein together with one or more additional active agents, e.g. a therapeutically active agent, as a combined preparation for separate, simultaneous or sequential use in the treatment or prevention of a disorder or condition associated with cytokine release from non-proliferating immune cells in blood, particularly a disorder or condition which results in or from, or is associated with, hypercytokinemia.

For instance, the invention provides a product or kit containing an agent as defined herein together with one or more additional active agents, e.g. a therapeutically active agent, as a combined preparation for separate, simultaneous or sequential use in the treatment or prevention of hypercytokinemia in a subject resulting from cytokine release from non-proliferating immune cells in blood.

Thus, an oligopeptidic compound (e.g. a peptide) capable of interacting with PCNA may contain or comprise a peptide motif (or sequence) that may be defined generally as:

X₁X₂X₃X₄X₅ (SEQ ID NO: 1), wherein:

X₁ is a basic amino acid;

X₂ is an aromatic amino acid;

X₃ is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P);

X₄ is any amino acid other than Proline (P), an acidic amino acid or an aromatic amino acid; and

X₅ is a basic amino acid or Proline (P).

In order that the oligopeptidic compound, which is capable of interacting with PCNA, or its encoding nucleic acid, may function in the methods and uses of the invention, the compound must be capable of entering the cell, i.e. crossing the cell membrane into the cytosol (cytoplasm), and optionally into one or more other cellular locations, e.g. the nucleus. This may be achieved using any convenient mechanism, e.g. associating directly or indirectly the oligopeptidic compound or its encoding nucleic acid molecule with one or more molecules capable of facilitating the uptake of said molecule into a cell, such as with a liposome or import peptide. A particularly advantageous mechanism may be to generate an oligopeptidic compound that comprises a domain that assists the transit of the compound across the cell membrane, i.e. to generate a fusion peptide or chimeric peptide (a peptide formed from two or more domains that are not normally found together in nature). For instance, a peptide comprising a cell membrane permeable motif, e.g. a cell penetrating peptide (an uptake or import peptide, or a peptide transduction domain). The fusion peptide (an oligopeptidic compound) may optionally comprise further sequences to facilitate the targeting of the peptide (i.e. to direct the peptide) to a particular sub-cellular location, e.g. a target peptide, signal peptide or transit peptide.

As the oligopeptidic compound comprises a PCNA interacting motif and a domain that facilitates its uptake, it will be evident that the compound comprises at least 5 residues and the final size of the compound will be dependent on the size and number of the domains that make up said compound, i.e. the PCNA interacting motif and uptake (import) peptide may be viewed as domains of the oligopeptidic compound. Thus, a domain may be viewed as a distinct portion (i.e. a sequence within the full-length peptidic sequence) of the oligopeptidic compound that can be assigned or ascribed a particular function or property.

In some embodiments, the oligopeptidic compound for use in the methods and uses of the invention comprises at least 2 domains, i.e. the PCNA interacting motif domain and the domain that facilitates the cellular uptake of said compound, e.g. uptake (import) peptide sequence domain. However, the oligopeptidic compound may comprise additional domains that may facilitate its function and/or stability, e.g. the capacity of the peptide to interact with its target, i.e. PCNA or an equivalent protein. Thus, the oligopeptidic compound may comprise at least 2, 3, 4 or 5 domains, e.g. 6, 7, 8, 9, 10, 12, 15 or more domains. For example, in some embodiments the oligopeptidic compound may comprise one or more linker domains, i.e. a domain that interspaces between two other domains, i.e. occupies the space in between and connects two domains of the oligopeptidic compound. In further embodiments, the oligopeptidic compound may comprise a domain that directs the oligopeptidic compound to a cellular or subcellular location, e.g. a signal peptide (also known as a target or transit peptide), such as a nuclear localization signal (NLS) sequence. In still further embodiments, the one or more linker domains may have an additional function, i.e. a linker domain may also function as a signal peptide, e.g. a NLS. Alternatively put, a signal peptide domain may function as a linker domain in some embodiments.

In an exemplary embodiment, the oligopeptidic compound may comprise a PCNA interacting motif domain, a domain that facilitates its cellular uptake (e.g. an uptake (import) peptide sequence domain) and a linker domain. In a further exemplary embodiment, the oligopeptidic compound may also comprise a nuclear localisation signal sequence domain. In still another embodiment the nuclear localization signal sequence domain may function as a linker domain.

Thus, it will be seen that in such embodiments the agent of the invention may take the form of a construct containing (i.e. comprising) an oligopeptidic compound which comprises a PCNA interacting motif as defined herein, together with a domain that facilitates its cellular uptake (e.g. an uptake peptide sequence) and optionally additional domains. In this aspect the invention may accordingly be seen to provide a construct comprising an oligopeptidic compound which is capable of interacting with PCNA.

As noted above the PCNA motif of the invention has been determined to mediate the interaction of an oligopeptidic compound (e.g. peptide) or protein containing such a motif with PCNA. Thus, the compound may be characterised insofar as it must be capable of interacting with PCNA, i.e. the oligopeptidic compounds for use in the methods and uses of the invention must be competent and/or proficient PCNA interacting molecules. The PCNA protein used to determine the capacity and/or affinity of the oligopeptidic compound:PCNA interaction may be from any suitable source, e.g. a PCNA from any animal, particularly a mammal such as a human, rodent (e.g. mouse, rat), canine or any other non-human animal. In preferred embodiments, the oligopeptidic compound:PCNA interaction is determined, characterised or assessed using human PCNA protein.

The interaction may be direct or indirect, and may involve direct binding of the motif to the PCNA protein, or the motif may bind indirectly, for example binding may be mediated by another molecule. This reference to “PCNA-interacting” or “PCNA-binding” can thus include any form of interaction, and both direct and indirect binding.

Any reference herein to a “motif” should be understood to mean X₁X₂X₃X₄X₅ as defined herein.

X₁ is preferably selected from lysine (K), arginine (R), histidine (H), ornithine (Orn), methyllysine (MeK), diaminobutyric acid (Dbu), citrulline (Cit), acetyllysine (AcK), and any suitable basic amino acid selected from the non-conventional amino acids in Table 2. Whilst the standard or conventional basic amino acids are preferred, e.g. K, R and H, particularly K and R, these may be substituted by any functionally equivalent non-conventional basic amino acid.

X₂ is preferably selected from phenylalanine (F), tryptophan (W), tyrosine (Y), tert.-butylglycine, cyclohexylalanine, tert.-butylphenylalanine, biphenylalanine and tri tert.-butyltryptophan (in certain embodiments this list may exclude F). Whilst the standard or conventional aromatic amino acids are preferred, e.g. F, W and Y, these may be substituted by any functionally equivalent non-conventional aromatic amino acid, e.g. from Table 2. In some embodiments, X₂ may be selected from W and Y, F and Y, or F and W or in specific embodiments X₂ may be F, or W or Y, or functionally equivalent non-conventional aromatic amino acids.

The binding of the motif to PCNA may in certain embodiments be improved when X₂ is W or Y. Thus, in one embodiment, X₂ is not F. However, as indicated above, in other embodiments it may be F.

X₃ is preferably a hydrophobic or polar amino acid, particularly an aliphatic amino acid or polar amino acid. Thus, in some embodiments, X₃ may be selected from leucine (L), isoleucine (I), valine (V), alanine (A) methionine (M), norleucine (Nor), serine (S), threonine (T), glutamine (Q), aspargine (N) or cysteine (C) or any suitable hydrophobic or polar amino acid selected from the non-conventional amino acids in Table 2. More particularly, X₃ may be selected from L, I, V, A, M, Nor, S or T and any suitable hydrophobic (preferably aliphatic) or polar (preferably a polar amino acid that does not contain an amine group (NH₂) in the R-group) amino acid selected from the non-conventional amino acids in Table 2. Preferably, X₃ is not N or Q or a non-conventional functional equivalent thereof and/or in certain embodiments X₃ is not M, S and/or T or a non-conventional functional equivalent thereof. X₃ may not be glycine (G) or proline (P) and this limitation is also intended to exclude non-conventional functional equivalents thereof.

Thus, in some embodiments, X₃ may be selected from L, I, A, V, M, S and T, and preferably from L, I, A, V, S and T and optionally non-conventional functional equivalents thereof.

In some embodiments, X₃ may be a hydrophobic, and more preferably an aliphatic amino acid. Thus, in some embodiments, X₃ may be selected from L, I, A, V, M, and preferably from L, I, V and A and optionally non-conventional functional equivalents thereof.

X₄ is preferably a hydrophobic, polar, basic or thiol-containing amino acid. Thus, in some embodiments X₄ an aliphatic amino acid or a polar amino acid. In some preferred embodiments, the polar amino acid does not contain an amine group (NH₂) in the R-group. Thus, X₄ preferably may be selected from L, I, V, A, M, Nor, S, T, Q, N, H, K, R, G or C and any suitable hydrophobic (preferably aliphatic) or polar (preferably a polar amino acid that does not contain an amine group (NH₂) in the R-group), basic or thiol-containing amino acid selected from the non-conventional amino acids in Table 2. Thus, in some embodiments, the basic amino acid may be selected from the amino acids as defined in X₁. In certain embodiments X₄ is not C or a non-conventional functional equivalent thereof and/or N or Q or a non-conventional functional equivalent thereof. In certain embodiments X₄ is not H and preferably X₄ is not R, K or H or a non-conventional functional equivalent thereof. In still further embodiments X₄ is not S or T or a non-conventional functional equivalent thereof. X₄ may not be P, an aromatic amino acid (as defined in X₂) or acidic amino acid, e.g. aspartic acid (D) or glutamic acid (E), and this limitation is also intended to exclude non-conventional functional equivalents thereof.

Thus, in some embodiments, X₄ may be selected from L, V, I, A, M, S, T and G, and preferably from L, V, A, I, S and T and optionally non-conventional functional equivalents thereof.

In other embodiments, X₄ may be a hydrophobic amino acid, and more preferably an aliphatic amino acid or G. Thus, in some embodiments, X₄ may be selected from L, I, A, V, M, and G and preferably from L, V, I and A and optionally non-conventional functional equivalents thereof.

X₅ is preferably selected from K, R, H, Orn, MeK, Dbu, Cit, AcK, P and any functionally equivalent amino acid selected from the non-conventional amino acids in Table 2. Whilst the standard or conventional amino acids are preferred, e.g. K, R, H and P, particularly K, R and H, e.g. K and R, these may be substituted by any functionally equivalent non-conventional basic amino acid.

Thus, in some embodiments X₅ is a basic amino acid, preferably selected from K, R and H and optionally non-conventional functional equivalents thereof.

Thus, in some embodiments X₃ and/or X₄ is a polar amino acid. Accordingly, in certain embodiments only one of X₃ and X₄ is a polar amino acid.

In some embodiments X₄ and/or X₅ is a basic amino acid. Accordingly, in certain embodiments X₅ is a basic amino acid.

A functionally equivalent amino acid may be defined as an amino acid that may be used as a substitute in a peptide or protein for a conventional amino acid without affecting significantly the function of the peptide or protein (or an amino acid that would not be expected to affect or alter significantly the function of the peptide or protein), e.g. an amino acid that has similar structural and/or chemical properties to the conventional amino acid. Thus, a functionally equivalent amino acid may be viewed as having the base structure of a standard amino acid, with a non-standard or non-conventional R-group that is structurally and/or chemically similar to the standard R-group. Preferably, the R-group is structurally similar to the standard R-group of the amino acid being substituted.

A conventional or standard amino acid is an amino acid that is used in vivo to produce a polypeptide or protein molecule, i.e. a proteinogenic amino acid. In other words, an amino acid with a standard or conventional R-group or an amino acid which possesses a side chain that is coded for by the standard genetic code, i.e. “coded amino acids”.

Thus, the invention may provide an oligopeptidic compound comprising the motif [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T/N/Q/C]-[L/I/V/A/M/G/S/T/N/Q/R/H/K/C]-[K/R/H/P] (SEQ ID NO: 2), wherein said oligopeptidic compound is capable of interacting with PCNA.

In another embodiment the motif may be defined as:

(SEQ ID NO: 3) [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T/N/Q]- [L/I/V/A/M/G/S/T/N/Q/R/H/K]-[K/R/H/P].

In another embodiment the motif may be defined as:

(SEQ ID NO: 4) [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T]- [L/I/V/A/M/G/S/T/N/Q/R/H/K]-[K/R/H/P].

In another embodiment the motif may be defined as:

(SEQ ID NO: 5) [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T]- [L/I/V/A/M/G/S/T/N/Q/R/H/K]-[K/R/H].

In another embodiment the motif may be defined as:

(SEQ ID NO: 6) [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T]- [L/I/V/A/M/G/S/T/R/K]-[K/R/H].

In another embodiment the motif may be defined as:

(SEQ ID NO: 7) [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T]-[L/I/V/A/M/G/S/T]- [K/R/H].

In another embodiment the motif may be defined as:

(SEQ ID NO: 8) [R/K]-[W/F/Y]-[L/I/V/A/M/S/T]-[L/I/V/A/M/G/S/T]- [K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 9) [R/K]-[W/F]-[L/I/V/A/M/S/T]-[L/I/V/A/M/G/S/T]- [K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 10) [R/K]-[W/F]-[L/I/V/A/M/T]-[L/I/V/A/M/G/S/T]-[K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 11) [R/K]-[W/F]-[L/I/V/A/M/T]-[L/I/V/A/M/S/T]-[K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 12) [R/K]-[W/F]-[L/I/V/A/M/S/T]-[L/I/V/A/M/G]-[K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 13) [R/K]-[W/F]-[L/I/A/V/M/T]-[L/I/V/A/M/S/T]-[K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 14) [R/K]-[W/F]-[L/I/V/A/M/S/T]-[L/V/A/I/S/T]-[K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 15) [R/K]-[W/F]-[L/V/I/A/T]-[L/V/A/I/S/T]-[K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 16) [R]-[W/F/Y]-[L/V/I/A]-[L/V/A/S/T/M]-[K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 17) [R]-[W/F/Y]-[L/V/I/A/T]-[L/V/A/S/T/M]-[K].

In another embodiment the motif may be defined as:

(SEQ ID NO: 18) [R/K]-[F/Y]-[L/V/I/A]-[L/V/A/I/M]-[K/R].

In another embodiment the motif may be defined as:

(SEQ ID NO: 19) [R/K]-[F/W/Y]-[L/I/V/A]-[L/I/V/A]-[K/R].

In yet another embodiment the motif may be defined as:

(SEQ ID NO: 20) [R/K]-[W/Y]-[L/V/I/A/S/T]-[L/V/A/S/T/M]-[K/R].

In yet another embodiment the motif may be defined as:

(SEQ ID NO: 21) [K]-[F/Y/W]-[L/V/I/A/T]-[L/V/A/I/S/T/]-[K].

In some embodiments X₁ and X₂ are RW, RF, KF, KW, RY or KY.

In some embodiments X₃ and X₄ are LL, LA, LV, AL, VL, VI, LI, IL, VV, VA, IV, II, AV, IA, AI, AM, LM, LS, LT, IS, MV, TV, AA, IM, LN, LQ, VM, TL, SL, IT, VT, LG, MA, ML, NL, QL, QI, TI, SI, AS, VS, SV, CA, IG, LR, VR, TK or IR. In some embodiments X₃ and X₄ are LL, LA, LV, AL, VL, VI, LI, IL, VV, VA, IV, II, AV, IA, AI, AM, LM, LS, LT, IS, MV, TV, AA, IM, LN, LQ or VM. In some embodiments X₃ and X₄ are LV, IV, SV, LS, AV, LG, LA, IR, LR, VR, AR, IK, LK, VK or AK. In particularly preferred embodiments X₃ and X₄ are LL, LA, LV, AL, VL, VI, LI, IL, VV, VA, IV, II, AV, IA, AI, AM, LM, LS or LT, preferably LL, LA, LV, AL, VL, VI, LI, IL, VV, VA, IV, II, AV, IA or AI. Thus, in certain embodiments X₃ and X₄ are not AG, AC, CC, NN, QQ, NQ, QN, TS, SS, ST or TT.

In some embodiments X₂ and X₃ are not FS or FT.

In some embodiments X₅ is K.

Thus, in a preferred embodiment, the oligopeptidic compound has or comprises the sequence RWLVK (SEQ ID NO: 28). In other preferred embodiments, the oligopeptidic compound has or comprises a sequence selected from any one or more of: RWLLK (SEQ ID NO: 22); RFLLK (SEQ ID NO: 23); RYLLK (SEQ ID NO: 24); RWLLR (SEQ ID NO: 25); RFLLR (SEQ ID NO: 26); RYLLR (SEQ ID NO: 27); RWLVK (SEQ ID NO: 28); RFLVK (SEQ ID NO: 29); RYLVK (SEQ ID NO: 30); RWLVR (SEQ ID NO: 31); RFLVR (SEQ ID NO: 32); RYLVR (SEQ ID NO: 33); RWIVK (SEQ ID NO: 34); RFIVK (SEQ ID NO: 35); RYIVK (SEQ ID NO: 36); RWIVR (SEQ ID NO: 37); RFIVR (SEQ ID NO: 38); RYIVR (SEQ ID NO: 39); RWLSK (SEQ ID NO: 40); RFLSK (SEQ ID NO: 41); RYLSK (SEQ ID NO: 42); RWLSR (SEQ ID NO: 43); RFLSR (SEQ ID NO: 44); RYLSR (SEQ ID NO: 45); RWISK (SEQ ID NO: 46); RFISK (SEQ ID NO: 47); RYISK (SEQ ID NO: 48); RWISR (SEQ ID NO: 49); RFISR (SEQ ID NO: 50); RYISR (SEQ ID NO: 51); RWSVK (SEQ ID NO: 52); RFSVK (SEQ ID NO: 53); RYSVK (SEQ ID NO: 54); RWSVR (SEQ ID NO: 55); RFSVR (SEQ ID NO: 56); RYSVR (SEQ ID NO: 57); RWAVK (SEQ ID NO: 58); RFAVK (SEQ ID NO: 59); RYAVK (SEQ ID NO: 60); RWAVR (SEQ ID NO: 61); RFAVR (SEQ ID NO: 62); RYAVR (SEQ ID NO: 63); RWLGR (SEQ ID NO: 64); RFLGR (SEQ ID NO: 65); RYLGR (SEQ ID NO: 66); RWLGK (SEQ ID NO: 67); RFLGK (SEQ ID NO: 68); RYLGK (SEQ ID NO: 69); RWLAR (SEQ ID NO: 70); RFLAR (SEQ ID NO: 71); RYLAR (SEQ ID NO: 72); RWLAK (SEQ ID NO: 73); RFLAK (SEQ ID NO: 74); RYLAK (SEQ ID NO: 75); RWLTK (SEQ ID NO: 76); RFLTK (SEQ ID NO: 77); RYLTK (SEQ ID NO: 78); RWLTR (SEQ ID NO: 79); RFLTR (SEQ ID NO: 80); RYLTR (SEQ ID NO: 81); RWITK (SEQ ID NO: 82); RFITK (SEQ ID NO: 83); RYITK (SEQ ID NO: 84); RWITR (SEQ ID NO: 85); RFITR (SEQ ID NO: 86); RYITR (SEQ ID NO: 87); RWTVK (SEQ ID NO: 88); RFTVK (SEQ ID NO: 89); RYTVK (SEQ ID NO: 90); RWTVR (SEQ ID NO: 91); RFTVR (SEQ ID NO: 92); RYTVR (SEQ ID NO: 93); RWIRK (SEQ ID NO: 94); RFIRK (SEQ ID NO: 95); RYIRK (SEQ ID NO: 96); RWIRR (SEQ ID NO: 97); RFIRR (SEQ ID NO: 98); RYIRR (SEQ ID NO: 99); RWLRK (SEQ ID NO: 100); RFLRK (SEQ ID NO: 101); RYLRK (SEQ ID NO: 102); RWLRR (SEQ ID NO: 103); RFLRR (SEQ ID NO: 104); RYLRR (SEQ ID NO: 105); KWLLK (SEQ ID NO: 106); KFLLK (SEQ ID NO: 107); KYLLK (SEQ ID NO: 108); KWLLR (SEQ ID NO: 109); KFLLR (SEQ ID NO: 110); KYLLR (SEQ ID NO: 111); KWLVK (SEQ ID NO: 112); KFLVK (SEQ ID NO: 113); KYLVK (SEQ ID NO: 114); KWLVR (SEQ ID NO: 115); KFLVR (SEQ ID NO: 116); KYLVR (SEQ ID NO: 117); KWIVK (SEQ ID NO: 118); KFIVK (SEQ ID NO: 119); KYIVK (SEQ ID NO: 120); KWIVR (SEQ ID NO: 121); KFIVR (SEQ ID NO: 122); KYIVR (SEQ ID NO: 123); KWLSK (SEQ ID NO: 124); KFLSK (SEQ ID NO: 125); KYLSK (SEQ ID NO: 126); KWLSR (SEQ ID NO: 127); KFLSR (SEQ ID NO: 128); KYLSR (SEQ ID NO: 129); KWISK (SEQ ID NO: 130); KFISK (SEQ ID NO: 131); KYISK (SEQ ID NO: 132); KWISR (SEQ ID NO: 133); KFISR (SEQ ID NO: 134); KYISR (SEQ ID NO: 135); KWSVK (SEQ ID NO: 136); KFSVK (SEQ ID NO: 137); KYSVK (SEQ ID NO: 138); KWSVR (SEQ ID NO: 139); KFSVR (SEQ ID NO: 140); KYSVR (SEQ ID NO: 141); KWAVK (SEQ ID NO: 142); KFAVK (SEQ ID NO: 143); KYAVK (SEQ ID NO: 144); KWAVR (SEQ ID NO: 145); KFAVR (SEQ ID NO: 146); KYAVR (SEQ ID NO: 147); KWLGR (SEQ ID NO: 148); KFLGR (SEQ ID NO: 149); KYLGR (SEQ ID NO: 150); KWLGK (SEQ ID NO: 151); KFLGK (SEQ ID NO: 152); KYLGK (SEQ ID NO: 153); KWLAR (SEQ ID NO: 154); KFLAR (SEQ ID NO: 155); KYLAR (SEQ ID NO: 156); KWLAK (SEQ ID NO: 157); KFLAK (SEQ ID NO: 158); KYLAK (SEQ ID NO: 159); KWLTK (SEQ ID NO: 160); KFLTK (SEQ ID NO: 161); KYLTK (SEQ ID NO: 162); KWLTR (SEQ ID NO: 163); KFLTR (SEQ ID NO: 164); KYLTR (SEQ ID NO: 165); KWITK (SEQ ID NO: 166); KFITK (SEQ ID NO: 167); KYITK (SEQ ID NO: 168); KWITR (SEQ ID NO: 169); KFITR (SEQ ID NO: 170); KYITR (SEQ ID NO: 171); KWTVK (SEC) ID NO: 172); KFTVK (SEC) ID NO: 173); KYTVK (SEQ ID NO: 174); KWTVR (SEQ ID NO: 175); KFTVR (SEQ ID NO: 176); KYTVR (SEQ ID NO: 177); KWLRK (SEC) ID NO: 178); KFLRK (SEQ ID NO: 179); KYLRK (SEQ ID NO: 180); KWLRR (SEQ ID NO: 181); KFLRR (SEC) ID NO: 182); KYLRR (SEQ ID NO: 183); KWIRK (SEC) ID NO: 184); KFIRK (SEC) ID NO: 185); KYIRK (SEC) ID NO: 186); KWIRR (SEC) ID NO: 187); KFIRR (SEQ ID NO: 188); KYIRR (SEC) ID NO: 189); RWVVK (SEC) ID NO: 190); RFVVK (SEC) ID NO: 191); RYVVK (SEQ ID NO: 192); RWVVR (SEC) ID NO: 193); RFVVR (SEQ ID NO: 194); RYVVR (SEC) ID NO: 195); KWVVK (SEQ ID NO: 196); KFVVK (SEC) ID NO: 197); KYVVK (SEQ ID NO: 198); KWVVR (SEC) ID NO: 199); KFVVR (SEC) ID NO: 200); KYVVR (SEQ ID NO: 201); RWALK (SEC) ID NO: 202); RFALK (SEQ ID NO: 203); RYALK (SEQ ID NO: 204); RWALR (SEQ ID NO: 205); RFALR (SEC) ID NO: 206); RYALR (SEQ ID NO: 207); KWALK (SEC) ID NO: 208); KFALK (SEQ ID NO: 209); KYALK (SEQ ID NO: 210); KWALR (SEC) ID NO: 211); KFALR (SEQ ID NO: 212); KYALR (SEQ ID NO: 213); RWVLK (SEC) ID NO: 214); RFVLK (SEQ ID NO: 215); RYVLK (SEQ ID NO: 216); RWVLR (SEQ ID NO: 217); RFVLR (SEC) ID NO: 218); RYVLR (SEQ ID NO: 219); KWVLK (SEC) ID NO: 220); KFVLK (SEQ ID NO: 221); KYVLK (SEQ ID NO: 222); KWVLR (SEC) ID NO: 223); KFVLR (SEQ ID NO: 224); KYVLR (SEQ ID NO: 225); RWILK (SEC) ID NO: 226); RFILK (SEC) ID NO: 227); RYILK (SEC) ID NO: 228); RWILR (SEQ ID NO: 229); RFILR (SEC) ID NO: 230); RYILR (SEC) ID NO: 231); KWILK (SEC) ID NO: 232); KFILK (SEC) ID NO: 233); KYILK (SEC) ID NO: 234); KWILR (SEC) ID NO: 235); KFILR (SEQ ID NO: 236); KYILR (SEC) ID NO: 237); RWVIK (SEC) ID NO: 238); RFVIK (SEC) ID NO: 239); RYVIK (SEC) ID NO: 240); RWVIR (SEC) ID NO: 241); RFVIR (SEQ ID NO: 242); RYVIR (SEC) ID NO: 243); KWVIK (SEQ ID NO: 244); KFVIK (SEC) ID NO: 245); KYVIK (SEC) ID NO: 246); KWVIR (SEC) ID NO: 247); KFVIR (SEC) ID NO: 248); KYVIR (SEC) ID NO: 249); RWIIK (SEQ ID NO: 250); RFIIK (SEQ ID NO: 251); RYIIK (SEC) ID NO: 252); RWIIR (SEC) ID NO: 253); RFIIR (SEC) ID NO: 254); RYIIR (SEC) ID NO: 255); KWIIK (SEC) ID NO: 256); KFIIK (SEC) ID NO: 257); KYIIK (SEC) ID NO: 258); KWIIR (SEQ ID NO: 259); KFIIR (SEQ ID NO: 260); KYIIR (SEC) ID NO: 261); RWLIK (SEC) ID NO: 262); RFLIK (SEQ ID NO: 263); RYLIK (SEC) ID NO: 264); RWLIR (SEQ ID NO: 265); RFLIR (SEC) ID NO: 266); RYLIR (SEC) ID NO: 267); KWLIK (SEC) ID NO: 268); KFLIK (SEC) ID NO: 269); KYLIK (SEC) ID NO: 270); KWLIR (SEC) ID NO: 271); KFLIR (SEQ ID NO: 272); KYLIR (SEC) ID NO: 273); RWIAK (SEC) ID NO: 274); RFIAK (SEC) ID NO: 275); RYIAK (SEC) ID NO: 276); RWIAR (SEQ ID NO: 277); RFIAR (SEQ ID NO: 278); RYIAR (SEQ ID NO: 279); KWIAK (SEQ ID NO: 280); KFIAK (SEQ ID NO: 281); KYIAK (SEQ ID NO: 282); KWIAR (SEQ ID NO: 283); KFIAR (SEQ ID NO: 284); KYIAR (SEQ ID NO: 285); RWVAK (SEQ ID NO: 286); RFVAK (SEQ ID NO: 287); RYVAK (SEQ ID NO: 288); RWVAR (SEQ ID NO: 289); RFVAR (SEQ ID NO: 290); RYVAR (SEQ ID NO: 291); KWVAK (SEQ ID NO: 292); KFVAK (SEQ ID NO: 293); KYVAK (SEQ ID NO: 294); KWVAR (SEQ ID NO: 295); KFVAR (SEQ ID NO: 296); KYVAR (SEQ ID NO: 297); and RWLVP (SEQ ID NO: 1209).

These specific sequences are listed by way of example and they are not intended to be limiting on the scope of the present invention. In some preferred embodiments the oligopeptidic compound has or comprises the sequence RWLVK (SEQ ID NO: 28) or KFIVK (SEQ ID NO: 119) or RWLVP (SEQ ID NO: 1209).

Whilst the PCNA interacting motifs listed above are preferred motifs of the invention, in some embodiments any one or more of these motifs may be excluded, e.g. any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more motifs may be excluded, such as any 25, 30, 40, 50 or more motifs or any integer in this range. Thus, in some embodiments the oligopeptidic compound does not have or comprise a sequence selected from any one or more of SEQ ID NOs: 22-297 and 1209.

Particular PCNA interacting motifs that may be excluded or disclaimed include any one or more of the following: KYMVR (SEQ ID NO: 298), KFLAK (SEQ ID NO: 158), KWLIK (SEQ ID NO: 268), KFLIK (SEQ ID NO: 269), KWLIOrn (SEQ ID NO: 299), KWLIDbu (SEQ ID NO: 300), and KWQLR (SEQ ID NO: 301).

The oligopeptidic compound is preferably an isolated compound, e.g. an isolated peptide and most preferably the oligopeptidic compound is a synthetic compound, e.g. a synthetic peptide. The nucleic acid molecule encoding the oligopeptidic compound is preferably an isolated nucleic acid molecule and most preferably the nucleic acid molecule is a synthetic nucleic acid molecule. In other words, the oligopeptidic compound and its encoding nucleic acid molecule are non-native, i.e. non-naturally occurring, molecules.

The domain that facilitates the uptake of the oligopeptidic compound may be an uptake (import) peptide sequence, which may be a sequence which acts to transport the oligopeptidic compound into a cell, or across a cell membrane (i.e. into the interior of a cell). It may thus be a so-called “cell penetrating” sequence (or more particularly “cell penetrating peptide”) also known in the art as a protein transduction domain (PTD) or protein transduction sequence.

Accordingly, as noted above the invention may provide an agent or construct comprising (i) an oligopeptidic compound comprising an APIM motif (i.e. a PCNA-interacting motif) as defined herein, and (ii) a cell penetrating sequence (more particularly a cell penetrating peptide).

Cell penetrating peptide (CPP) technology has developed greatly over recent years and a wide variety of cell penetrating peptides are known and described in the art and indeed a range of such peptides are commercially available. Cell penetrating peptides may vary greatly in size, sequence and charge, and indeed in their mechanism of function (which is presently not known for some peptides and not fully elucidated for others), but share the common ability to translocate across the plasma membrane and deliver an attached or associated moiety (the so-called “cargo”) into the cytoplasm of a cell. CPPs are thus peptide-based delivery vectors.

Whilst CPPs are not characterized by a single structural or functional motif, tools to identify CPPs are available and the skilled person can readily determine whether a peptide sequence may function to facilitate the uptake of the peptide of which it forms a domain, i.e. whether a domain may function as an uptake (import) peptide, e.g. a CPP. For example, Hansen et al (Predicting cell-penetrating peptides, Advanced Drug Delivery Reviews, 2008, 60, pp. 572-579), provides a review of methods for CPP prediction based on the use of principal component analysis (“z-predictors”) and corresponding algorithms based on original work by Hällbrink et al (Prediction of Cell-Penetrating Peptides, International Journal of Peptide Research and Therapeutics, 2005, 11(4), pp. 249-259). In brief, the methodology works by computing z-scores of a candidate peptide as based on a numerical value and an associate range. If the z-scores fall within the range of known CPP z-scores, the examined peptides are classified as CPPs. The method was shown to have high accuracy (about 95% prediction of known CPPs).

Additional methods for the prediction of CPPs have been developed subsequently (see e.g. Sanders et al., Prediction of Cell Penetrating Peptides by Support Vector Machines, PLOS Computational Biology, 2011, 7(7), pp. 1-12, herein incorporated by reference) and a CPP database is available (Gautam et al., CPPSite: a curated database of cell penetrating peptides, Database, 2012, Article ID bas015 and crdd.osdd.net/raghava/cppsite, both herein incorporated by reference). Accordingly, any suitable CPP may find utility in the invention and, as discussed below, a variety of CPPs have already been identified and tested and could form the basis for determining and identifying new CPPs.

CPPs may be derived from naturally-occurring proteins which are able to translocate across cell membranes such as the Drosophila homeobox protein Antennapedia (a transcriptional factor), viral proteins such as the HIV-1 transcriptional factor TAT and the capsid protein VP22 from HSV-1, and/or they may be synthetically-derived, e.g. from chimeric proteins or synthetic polypeptides such as polyarginine. As noted above, there is not a single mechanism responsible for the transduction effect and hence the design of CPPs may be based on different structures and sequences. Cell penetrating peptides are also reviewed in Jarver et al. 2006 (Biochimica et Biophysica Acta 1758, pages 260-263) and Table 1 below lists various representative peptides. U.S. Pat. No. 6,645,501 (herein incorporated by reference) further describes various cell penetrating peptides which might be used.

TABLE 1 CPP SEQUENCE REFERENCE Antp Class Penetratin RQIKIWFQNRRMKWKK (SEQ ID NO: 302) Bolton (2000) Eur. J. Neuro. 12:287 Penatratin RRMKWKK (SEQ ID NO: 303) U.S. Pat. No. derivatives NRRMKWKK (SEQ ID NO: 304) 6,472,507 QNRRMKWKK (SEQ ID NO: 305) EP4855781 FQNRRMKWKK (SEQ ID NO: 306) WO 97/12912 RREKWKK (SEQ ID NO: 307) RRQKWKK (SEQ ID NO: 308) KRMKWKK (SEQ ID NO: 309) RKMKWKK (SEQ ID NO: 310) RROKWKK (SEQ ID NO: 311) RRMKQKK (SEQ ID NO: 312) RRMKWFK (SEQ ID NO: 313) RORKWKK (SEQ ID NO: 314) RRMWKKK (SEQ ID NO: 315) RRMKKWK JSEQ ID NO: 316) (using standard single amino acid notation, ornithine (O), diaminobutyric acid (B),  norleucine (N)) D-Penetratin rqikiwfqnrrmkwkk (SEQ ID NO: 317) Rouselle, C. et al. (2000) Mol. Pharm 57:679 Protegrin Class Pegelin RGGRLSYSRRRFSTSTGR (SEQ ID NO: 318) Rouselle, C. et (SynB) al. (2000) Mol. Pharm 57:679 HIV-TAT Class HIV-TAT GRKKRRQRRRPPQ (SEQ ID NO: 319) Vives E.J Biol, Chem 1997, 272:16010 Snyder (2004) PLOS 2: 186 47-57 OF YGRKKRRQRRR (SEQ ID NO: 320) Potocky et al. HIV-TAT (2003) JBC VP22 DAATATRGRSAASRPTERPRAPARSASRPRRVD Elliott g. Cell (SEQ ID NO: 321) 1997, 88:223- 233 Amphipathic peptides MAP KLALKLALKALKAALKLA (SEQ ID NO: 322) Morris MC., Nat Biotechnol. 2001, 19:1173-1176 Transportan GWTLNSAGYLLGKINLKALAALAKKIL Pooga M, (SEQ ID NO: 323) FASEB J 1998, 12:67- 77 Transportan- AGYLLGKINLKALAALAKKIL (SEQ ID NO: 324) Soomets U, 10 Biochim Biophys Acta 2000, 1467:165-176 KALA WEAKLAKALAKALAKHLAKALAKALKACEA  Oehike J., (SEQ ID NO: 325) Biochim Biophys Acta 1998, 1414:127-139 Pep-1 KETWWETWWTEWSQPKKKRKV Wyman (SEQ ID NO: 326) Biochemistry 1997, 36:3008-3017 Pep-2 KETWFETWFTEWSQPKKKRKV (SEQ ID NO: 327) MPG GALFLGFLGAAGSTMGAWSQPKSKRKV Wagstaff KM (SEQ ID NO: 328) Curr Med Chem 2006, 13:1371-1387 Vectocell VKRGLKLRHVRPRVTRMDV (SEQ ID NO: 329) Coupade peptides SRRARRSPRHLGSG* (SEQ ID NO: 330) (2005) LRRERQSRLRRERQSR* (SEQ ID NO: 331) Biochem. J. GAYDLRRRERQSRLRRRERQSR 407 (SEQ ID NO: 332) *indicates addition of cys for conjugation to cargo Wr-T KETWWETWWTEWWTEWSQ-GPG-rrrrrrrr Kondo (2004) transporter (SEQ ID NO: 333) Mol. Can. r = D-enantiomer arginine Thera 1623 Other peptides R7 RRRRRRR (SEQ ID NO: 334) Rothbard et al., Nat. Med 6 (2000) 1253- 1257

Antennapedia-derived CPPs (Antp class) represent a class of particular interest, based around the 16 amino acid Penetratin sequence as shown in Table 1, which corresponds to the third loop of antennapedia protein and was shown to be responsible for translocation of the protein. Penetratin has been extensively developed as a delivery vehicle, including particularly for pharmaceutical use, and a wide range of Penetratin derivatives and modified sequences have been proposed and described. Reference may be made in particular to WO 91/1891, WO 00/1417, WO 00/29427, WO 2004/069279 and U.S. Pat. No. 6,080,724 (herein incorporated by reference). Thus, the 16 amino acid sequence of Penetratin may be modified and/or truncated, or the peptide may be chemically-modified or retro-, inverso- or retro-inverso analogues may be made whilst retaining cell-penetrating activity.

Another group of cell penetrating peptides which may advantageously be used are based on the HIV-TAT sequence and HIV-TAT and fragments thereof represent a preferred class of CPPs for use according to the present invention. Various TAT-based CPPs are described in U.S. Pat. No. 5,656,122 (herein incorporated by reference). An exemplary HIV-TAT peptide as used in the Examples below is RKKRRQRRR (SEQ ID NO: 335) but it will readily be appreciated that longer or shorter TAT fragments may be used.

As mentioned above no particular structural features or sequence motifs are common to all CPPs. However, various classes of CPPs may be identified by particular features, such as for example peptides which are amphipathic and net positively charged. Other groups of CPPs may have a structure exhibiting high α-helical content. Another group may be peptides characterised by a high content of basic amino acids. CPPs may thus be or may comprise oligomers of basic amino acids such as arginine e.g. 5 to 20, 6 to 15 or 6 to 12 R residues e.g. R₇ (SEQ ID NO: 334), R₈ (SEQ ID NO: 336) or R₁₁(SEQ ID NO: 337) or QSR₈ (SEQ ID NO: 338).

Proline-rich amphipathic peptides are another class of CPP and such peptides characterised by the presence of pyrrolidine rings from prolines are described in Pujals et al. 2008 Advanced Drug Delivery Reviews 60, pages 473-484 (herein incorporated by reference).

Other successfully developed CPPs include pVEC (Elmquist et al. 2003 Biol. Chem 384, pages 387-393; Holm et al. 2005 Febs Lett. 579, pages 5217-5222, all herein incorporated by reference) and calcitonin-derived peptides (Krauss et al. 2004 Bioorg. Med. Chem. Lett., 14, pages 51-54 herein incorporated by reference).

Commercially available CPPs include Chariot, based on the Pep-1 peptide (Active Motif, France), the Syn-B vectors based on the protegrin peptide PG-1 (Syntem, France), and Express-si Delivery based on the MPG peptide from Genospectra, USA.

Other CPPs include the R41, R8, M918 and YTA-4 peptides (SEQ ID NOs: 1210-1213, respectively) disclosed in Eriksson et al. 2013, Antimicrobial Agents and Chemotherapy, vol. 57(8), pp. 3704-3712 (incorporated herein by reference).

In some embodiments the CPPs may be cyclic peptides, such as those disclosed in Oh et al., 2014, Mol. Pharmaceutics, vol. 11, pp. 3528-3536 (incorporated herein by reference). In particular, the CPPs may be amphiphilic cyclic CPPs, particularly containing tryptophan and arginine residues. In some embodiments the CPPs may be cyclic polyarginine peptides and may be modified by the addition of a fatty acyl moiety, e.g. octanoyl, dodecanoyl, hexadecanoyl, N-acetyl-L-tryptophanyl-12-aminododecanoyl etc. Suitable cyclic CPPs for use in the invention are presented in SEQ ID NOs: 1214-1220.

In addition to publically available and reported CPPs, novel or derivative CPP peptides may be designed and synthesized based on known or reported criteria (e.g. known CPP sequences or features such as basic amino acid content, α-helical content etc. as discussed above). Additionally, randomly-designed or other peptides may be screened for CPP activity, for example by coupling or attaching such a peptide containing a reporter molecule, e.g. a detectable label or tag such as a fluorescent tag to the desired cargo (e.g. an oligopeptidic compound as described herein) and testing to see if the construct is translocated across the cell membrane, for example by adding these peptides to live cells followed by examination of cellular import e.g. using confocal microscopy.

Indeed, whilst it is generally the case that a CPP will penetrate or enter virtually any cell type, it may in some cases be observed that successful or efficient delivery may be dependent, or may vary depending, on the precise nature of the cargo (e.g. cargo peptide sequence) and/or the CPP used. It would be well within the routine skill of the person skilled in the art to determine optimum peptide sequences and combinations etc, and to test and/or modify cargo and/or CPP sequence or structure etc.

Thus, by way of summary, the skilled person will be aware of suitable peptide sequences that may facilitate the uptake of the oligopeptidic compound, but by way of example the sequences may include Penetratin™, a 16-amino acid peptide corresponding to the third helix of the homeodomain of Antennapedia protein, R rich tags such as R6-Penetratin (in which arginine-residues were added to the N-terminus of Penetratin) and derivatives of the HIV Tat protein such as GRKKRRQRRRPPQQ (SEQ ID NO: 339).

Thus, in some embodiments the domain that facilitates the cellular uptake of the oligopeptidic compound is a CPP and may be selected from any one of:

(i) an antennapedia class peptide;

(ii) a protegrin class peptide;

(iii) a HIV-TAT class peptide;

(iv) an amphipathic class peptide selected from an amphipathic and net positively charged peptide, a proline-rich amphipathic peptide, a peptide based on the Pep-1 peptide and a peptide based on the MPG peptide;

(v) a peptide exhibiting high α-helical content;

(vi) a peptide comprising oligomers of basic amino acids;

(vii) pVEC;

(viii) a calcitonin-derived peptide; and

(ix) an amphiphilic cyclic CPP.

In some embodiments the domain that facilitates the cellular uptake of the oligopeptidic compound is a CPP and may be selected from a sequence selected from any one of SEQ ID NOs: 302-1162 or a fragment and/or derivative thereof. The details and properties of the CPPs identified in SEQ ID NOs: 340-1162 can be found at crdd.osdd.net/raghava/cppsite, CPPSite: A database of cell penetrating peptides (herein incorporated by reference).

In some embodiments the domain that facilitates the cellular uptake of the oligopeptidic compound is SEQ ID NO: 337.

In some embodiments, the oligopeptidic compound also comprises one or more domains that provide a signal (target or transit) sequence. In some embodiments, the signal sequence may target the oligopeptidic compound to a specific cell type. Additionally or alternatively, in some embodiments the oligopeptidic compound may comprise a signal peptide that localises the compound to a specific intracellular compartment, e.g. the nucleus. In some embodiments, the oligopeptidic compound is targeted to the cytosol, which may be achieved without an additional signal peptide, i.e. the uptake (import) peptide, e.g. CPP, may be sufficient to direct or localise the oligopeptidic compound to the cytosol of a cell.

The signal sequence or signal sequence domain may thus be viewed as any sequence which acts to localise, or alternatively put, to direct, translocate or transport, the oligopeptidic compound to any desired location e.g. to any desired cell type or subcellular location, e.g. nucleus.

As mentioned above, the oligopeptidic compound (or constructs) for use in the use and methods of the invention may comprise one or more signal sequences (i.e. one or more domains that function as signal sequences), e.g. a signal peptide which directs the compound (or construct) into a particular sub-cellular compartment, such as the nucleus. Nuclear localisation signals (NLSs) are again well known in the art and widely described in the literature. For instance, a searchable database of known and predicted NLSs is available, see e.g. Cokol et al (Finding nuclear localization signals, EMBO Reports, 2000, 1(5), pp. 411-415, herein incorporated by reference). The PSORT II database, psort.hgc.jp (herein incorporated by reference) can be used for the prediction of nuclear localization of proteins based on NLSs. Accordingly, any known or functional NLS may find utility in the invention.

An NLS may vary in length and/or sequence and a wide range of specific NLS sequences have been described. In general, however, it has been found that peptides comprising positively charged amino acids (notably lysine (K), arginine (R) and/or histidine (H)) may function as an NLS. An exemplary NLS may thus be a peptide of e.g. 4-20, more particularly 4-15, 4-12, 4-10 or 4-8 amino acids, wherein at least 4 amino acids (and more particularly at least 60, 70, 75, 80, 85, or 90% of the amino acid residues in the NLS peptide) are positively charged amino acids, preferably selected from K, R or H. Such an exemplary NLS may for example have or comprise the sequence RKRH (SEQ ID NO: 1163).

Nuclear localisation signals, including both actual experimentally-determined and predicted or proposed NLS sequences, and strategies for identifying NLSs are also described in Lange et al., J. Biol. Chem. 2007, 282(8), 5101-5105; Makkerh et al., Current Biology 1996, 6(8), 1025-1027; Leslie et al., Methods 2006, 39, 291-308; and Lusk et al. Nature Reviews MCB 2007, 8, 414-420 (all herein incorporated by reference).

A classical NLS consists of either one (monopartite) or two (bipartite) stretches of basic amino acids. A monopartite NLS may be exemplified by the SV40 large T antigen NLS (¹²⁶PKKKRKV¹³² [SEQ ID NO: 1164]) and a bipartite NLS by the nucleoplasmin NLS (¹⁵⁵ KRPAATKKAGQAKKKK¹⁷⁰ [SEQ ID NO: 1165]). The monopartite NLS consensus sequence K-[K/R]-X-[K/R] (SEQ ID NO: 1166) has been proposed and accordingly an NLS according to the present invention may in one embodiment comprise or consist of such a consensus sequence (where X is any amino acid).

A representative bipartite NLS according to the invention may have the sequence KR-[X]₅₋₂₀-KKKK (SEQ ID NO: 1167), e.g. KR-X₁₀-KKKK (SEQ ID NO: 1168) (where X is any amino acid).

An alternative exemplary bipartite NLS may take the form RKRH-[X]₂₋₁₀-KK (SEQ ID NO: 1169) e.g. RKRH-X₂-KK (SEQ ID NO: 1170), for example RKRH-II-KK (SEQ ID NO: 1171).

The oncoprotein c-myc NLS differs from classical NLSs in that only 3 of 9 amino acid residues are basic (PAAKRVKLD [SEQ ID NO: 1172]), indicating that an NLS need not necessarily conform to the consensus or classical sequences given above. Makkerh et al (supra) describe NLS sequences in which a cluster of basic amino acids (e.g. KKKK [SEQ ID NO: 1173]) is flanked by neutral and acidic residues, for example PAAKKKKLD (SEQ ID NO: 1174).

Other possible NLS sequences which may be given by way of example include: PKKKRKVL (SEQ ID NO: 1175), KKKRK (SEQ ID NO: 1176), KKKRVK (SEQ ID NO: 1177), KKKRKVL (SEQ ID NO: 1178) and RKKRKVL (SEQ ID NO: 1179). Any NLS which is a derivative of a known NLS e.g. the SV40, nucleoplasmin, UNG2 or c-myc NLS may be used.

A putative, proposed or predicted NLS sequence can be tested for NLS activity using principles and assays known and described in the art. For example, a candidate NLS sequence may be attached to the desired cargo (in this case an oligopeptidic compound as defined herein) and the construct may be provided with a detectable reporter molecule (e.g. a tag or label which may be visualised, for example a fluorescent label) and contacted with a test cell. Distribution of the construct in the cell may then be determined.

Thus, by way of summary, the skilled person will be aware of suitable signal sequences, but by way of example the following are mentioned herein Examples of nuclear localisation sequences include the SV40 protein derivative KKKRK (SEQ ID NO: 1176).

Thus, in some embodiments the oligopeptidic compound comprises a signal sequence (i.e. a domain comprising a signal peptide) that localizes or directs the oligopeptidic compound to a sub-cellular location, such as a NLS and may be selected from any one of:

(i) a peptide of 4-20 amino acids, wherein at least 4 amino acids are positively charged amino acids, preferably selected from K, R or H; and/or

(ii) a sequence selected from any one of SEQ ID NOs: 1163-1179 or a fragment and/or derivative thereof.

In some embodiments the nuclear localisation signal sequence comprises a sequence selected from any one of SEQ ID NOs: 1163-1179 or a fragment and/or derivative thereof, preferably wherein said fragment and/or derivative comprises at least 4 positively charged amino acids, preferably selected from any of K, R or H.

In some embodiments an oligopeptidic compound or construct according to the present invention may comprise at least three domains, including (i) an APIM motif domain as defined herein, (ii) a linker domain, which may in some embodiments comprise a nuclear localisation signal sequence, and (iii) a peptide sequence domain that facilitates the cellular uptake of said compound or construct (i.e. an uptake/import peptide sequence domain, e.g. cell penetrating signal sequence domain).

The separate elements or components (domains) of a construct according to the present invention may be contained or presented in any order, but preferably in the orders indicated above (e.g. APIM oligopeptidic compound-CPP or APIM oligopeptidic compound-linker-CPP).

In some embodiments, the APIM motif is located at or towards the N-terminus of the peptide. For instance, the APIM motif may be described as being N-terminal to the peptide sequence domain that facilitates the cellular uptake of said compound (e.g. the CPP) and optionally N-terminal to the linker sequence, if present.

In a preferred embodiment, the oligopeptidic compound comprises a PCNA interacting motif as set forth in SEQ ID NO: 28, a nuclear localisation signal sequence as set forth in SEQ ID NO: 1176 and a cell penetrating signal sequence as set forth in SEQ ID NO: 337.

Furthermore, in some embodiments an oligopeptidic compound or construct according to the invention may contain more than one PCNA-interacting motif. Thus, alternatively put, an agent for use in the uses and methods of the present invention may contain or encode an oligopeptidic compound comprising more than one PCNA-interacting motif. A construct or oligopeptidic compound may for example contain 1-10, e.g. 1-6, or 1-4 or 1-3 or one or two motifs. Within a construct also containing a signal sequence, such motifs may be spaced or located according to choice, e.g. they may be grouped together, or they may be separated by signal sequence elements e.g. motif-motif-CPP, motif-linker-motif-CPP; or motif-linker-motif-motif-CPP; or motif-motif-linker-CPP etc.

As referred to herein a “fragment” may comprise at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or 99% of the amino acids of the sequence from which it is derived. Said fragment may be obtained from a central or N-terminal or C-terminal portions of the sequence. Whilst the size of the fragment will depend on the size of the original sequence, in some embodiments the fragments may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid residues shorter than the sequence from which it is derived, e.g. 1-10, 2-9, 3-8, 4-7 amino acid residues shorter than the sequence from which it is derived.

As referred to herein a “derivative” of a sequence is at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to the sequence to which it is compared.

Sequence identity may be determined by, e.g. using the SWISS-PROT protein sequence databank using FASTA pep-cmp with a variable pamfactor, and gap creation penalty set at 12.0 and gap extension penalty set at 4.0, and a window of 2 amino acids. Preferably said comparison is made over the full length of the sequence, but may be made over a smaller window of comparison, e.g. less than 200, 100, 50, 20 or 10 contiguous amino acids.

Preferably such sequence identity related polypeptides, i.e. derivatives, are functionally equivalent to the peptides which are set forth in the recited SEQ ID NOs. Similarly, the peptides with sequences as set forth in the SEQ ID NOs. may be modified without affecting the sequence of the polypeptide as described below.

Furthermore, “fragments” as described herein may be functional equivalents. Preferably these fragments satisfy the identity (relative to a comparable region) conditions mentioned herein.

As referred to herein, to achieve “functional equivalence” the peptide may show some reduced efficacy in performing the function relative to the parent molecule (i.e. the molecule from which it was derived, e.g. by amino acid substitution), but preferably is as efficient or is more efficient. Thus, functional equivalence may relate to a peptide which is effective in localizing or directing the oligopeptidic compound to the cell type or cellular location, e.g. to facilitate to the uptake of the peptide as described above. This may be tested by comparison of the effects of the derivative peptide relative to the peptide from which it is derived in a qualitative or quantitative manner, e.g. by performing the in vitro analyses described above. Where quantitative results are possible, the derivative is at least 30, 50, 70 or 90% as effective as the parent peptide.

Functionally-equivalent peptides which are related to or derived from the parent peptide, may be obtained by modifying the parent amino acid sequence by single or multiple amino acid substitution, addition and/or deletion (providing they satisfy the above-mentioned sequence identity requirements), but without destroying the molecule's function. Preferably the parent sequence has less than 20 substitutions, additions or deletions, e.g. less than 10, 5, 4, 3, 2, or 1 such modifications. Such peptides may be encoded by “functionally-equivalent nucleic acid molecules” which may be generated by appropriate substitution, addition and/or deletion of one or more bases.

The domains (which may be viewed as components, elements or separate parts) of an oligopeptidic compound or construct as described herein may be attached or linked to one another in any desired or convenient way according to techniques well known in the art. Thus, the domains may be linked or conjugated chemically, e.g. using known chemical coupling technologies or the compound or constructs may be formed as a single whole using genetic engineering techniques e.g. techniques for forming fusion proteins, or they may simply be synthesized as a whole, e.g. using peptide synthesis techniques.

The domains may be linked directly to each other or they may be linked indirectly by means of one or more linker (or spacer) sequences. Thus, a linker sequence may interspace or separate two or more individual domains (i.e. parts, e.g. or separate motif elements) in an oligopeptidic construct or compound. The precise nature of the linker sequence is not critical and it may be of variable length and/or sequence, for example it may have 0-40, more particularly 0-20, 0-15, 0-12, 0-10, 0-8, or 0-6, 0-4 or 0-3 residues e.g. 1, 2 or 3 or more residues. By way of representative example the linker sequence, if present, may have 1-15, 1-12, 1-10, 1-8, 1-6 or 1-4 residues etc. The nature of the residues is not critical and they may for example be any amino acid, e.g. a neutral amino acid, or an aliphatic amino acid, or alternatively they may be hydrophobic, or polar or charged or structure-forming e.g. proline. A range of different linker sequences have been shown to be of use, including short (e.g. 1-6) sequences of neutral and/or aliphatic amino acids.

Exemplary linker sequences thus include any single amino acid residue, e.g. A, I, L, V, G, R, Q, T, or W, or a di-, tri- tetra- penta- or hexa-peptide composed of such residues.

As representative linkers may be mentioned I, II, IL, R, W, WW, WWW, RIL, RIW, GAQ, GAW, VAT, IILVI (SEQ ID NO: 1180), IILVIII (SEQ ID NO: 1181) etc.

The linkers between different domains (components, elements or parts) may be the same or different.

As mentioned above, in some embodiments the linker may comprise or consist of an NLS. Alternatively viewed, in some embodiments an NLS, when present, may function both as a signal peptide and a linker. Thus, the oligopeptidic compound may comprise a signal peptide (e.g. an NLS) and a linker.

Representative compounds (or more particularly constructs) for use in the methods and uses of the invention include:

(SEQ ID NO: 1182) MDRWLVKRILVATK, (SEQ ID NO: 1183) MDRWLVKRILKKKRKVATKG, (SEQ ID NO: 1184) MDRWLVKGAQPKKKRKVLRQIKIWFQNRRMKWKK, (SEQ ID NO: 1185) MDRWLVKGAWKKKRVKIIRKKRRQRRRK, (SEQ ID NO: 1186) MDRWLVKGAWKKKRKIIRKKRRQRRRG, (SEQ ID NO: 1187) MDRWLVKGAWKKKRKIIRKKRRQRRRK, (SEQ ID NO: 1188) MDRWLVKRIWKKKRKIIRKKRRQRRRK, (SEQ ID NO: 1189) MDRWLVKWWWKKKRKIIRKKRRQRRRK, (SEQ ID NO: 1190) MDRWLVKWWRKRHIIKKRKKRRQRRRK, (SEQ ID NO: 1191) MDRWLVKRIWKKKRKIIRRRRRRRRRRRK, (SEQ ID NO: 1192) MDRWLVKRIWKKKRKIIRQIKIWFQNRRMKWKK, (SEQ ID NO: 1193) MDRFLVKGAWRKRHIIKKRKKRRQRRRK, (SEQ ID NO: 1194) MDRWLVKWKKKRKIRRRRRRRRRRRK, (SEQ ID NO: 1195) MDRWLVKWKKKRKIRKKRRQRRRK, (SEQ ID NO: 1196) MDRWLVKWRKRHIRKKRRQRRRK, (SEQ ID NO: 1197) Ac-MDRWLVKGAWRKRHIRKKRRQRRRK, (SEQ ID NO: 1198) Ac-MDRWLVKWKKKRKIRRRRRRRRRRR, (SEQ ID NO: 1199) Ac-MDRFLVKWKKKRKIRRRRRRRRRRR, (SEQ ID NO: 1200) Ac-MDRWLVKKKKRKRRRRRRRRRRRK, (SEQ ID NO: 1201) Ac-MDRWLVKKKKRKRRRRRRRRRRR, (SEQ ID NO: 1202) MDRWLVKRIWKKKRKIIRWLVKWWWRKKRRQRRRK, (SEQ ID NO: 1203) MDRWSVKWKKKRKIRRRRRRRRRRR  (SEQ ID NO: 1204) MDRWAVKWKKKRKIRRRRRRRRRRR  or (SEQ ID NO: 1208) MDRWLVPWKKKRKIRRRRRRRRRRR.

In a particularly preferred embodiment, the oligopeptidic compound comprises a sequence as set forth in SEQ ID NO: 1198, 1203, 1204 or 1208.

The oligopeptidic compounds shown above comprise N-terminal amino acids that do not form part of the domains that are essential for the compounds to have activity in the methods and uses of the invention, i.e. an “MD” sequence. Some of the peptides also comprise N-terminal modification, e.g. acetyl groups. These additional amino acids and modifications may facilitate the production of the oligopeptidic compounds, e.g. in vitro or in vivo, and/or help to protect the compounds from degradation in vivo. It will be evident that the oligopeptidic compounds do not require these additional amino acids or modifications for their activity. Accordingly, further representative sequences according to the invention include any of SEQ ID NOs: 1182 to 1204 or 1208, omitting the N-terminal “MD” and/or “Ac” groups. In other embodiments, a C-terminal K or G residue may additionally or alternatively be omitted. Furthermore, the presence of additional amino acids or modifications at either terminus would not be expected to disrupt or inhibit the function of the oligopeptidic compounds described herein. Thus, in some embodiments, the oligopeptidic compound may comprise an N-terminal sequence, e.g. a sequence at the N-terminus that does not comprise a domain defined above, e.g. a so-called N-terminal flanking sequence. In some embodiments, the oligopeptidic compound may comprise a C-terminal sequence, e.g. a sequence at the C-terminus that does not comprise a domain defined above, e.g. a so-called C-terminal flanking sequence. In some embodiments, the oligopeptidic compound may comprise an N-terminal and C-terminal flanking sequence.

A flanking sequence may comprise from about 1-150 amino acids, such as 1-120, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-35, 1-30 etc. Thus, a flanking sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids, e.g. 1-40, 2-39, 3-38, 4-37, 5-36, 6-35, 7-34, 8-33, 9-32, 10-31, 11-30, 12-29, 13-28, 14-27, 15-26 amino acids or any combination thereof.

Oligopeptidic compounds having sequences as set out in SEQ ID NOs. 1182-1204 and 1208 comprise separate domains (i.e. components) making up the constructs (i.e. motif-containing sequence, linker/NLS, CPP, etc.) Thus, it will be seen that SEQ ID NOs. 1182-1204 and 1208 represent constructs comprising at least one motif-containing sequence, a linker/NLS and a CPP, in some cases linked by linker sequences which may vary in sequence, as specified. NLS sequences based on the SV40 or UNG2 NLS sequences are used, and CPP sequences based on Penetratin, HIV-TAT or an R-rich peptide.

The standard amino acid one letter code is used herein, so K stands for lysine (Lys), I stands for isoleucine (Ile) and so on.

As mentioned above, the oligopeptidic compound, and more particularly, the APIM motif, may comprise non-conventional or non-standard amino acids. Other domains in the oligopeptidic compound may also incorporate non-standard amino acids. In some embodiments, the oligopeptidic compound may comprise one or more, e.g. at least 1, 2, 3, 4 or 5 non-conventional amino acids, i.e. amino acids which possess a side chain that is not coded for by the standard genetic code, termed herein “non-coded amino acids” (see e.g. Table 2). These may be selected from amino acids which are formed through metabolic processes such as ornithine or taurine, and/or artificially modified amino acids such as 9H-fluoren-9-ylmethoxycarbonyl (Fmoc), (tert)-(B)utyl (o)xy (c)arbonyl (Boc), 2,2,5,7,8-pentamethylchroman-6-sulphonyl (Pmc) protected amino acids, or amino acids having the benzyloxy-carbonyl (Z) group. Preferably, where such non-coded amino acids are present, they are not located within the motif, but in some embodiments one or more non-coded amino acids are present within the motif. In some embodiments, non-coded amino acids are present in more than one domain of the oligopeptidic compound.

In vitro and/or in vivo stability of the oligopeptidic compound may be improved or enhanced through the use of stabilising or protecting means known in the art, for example the addition of protecting or stabilising groups, incorporation of amino acid derivatives or analogues or chemical modification of amino acids. Such protecting or stabilising groups may for example be added at the N and/or C-terminus. An example of such a group is an acetyl group and other protecting groups or groups which might stabilise a peptide are known in the art.

The oligopeptidic compounds of the invention will typically comprise only amino acids having the L-configuration, but one or more amino acids having the D configuration may be present. In some embodiments the oligopeptidic compound contains at least 1, 2, 3, 4 or 5 D-amino acids and they are preferably found in the motif, but in another embodiment, D-amino acids are present only outside of the motif. In a still further embodiments, D-amino acids may be found in more than one domain of the oligopeptidic compound. The oligopeptidic compound may be linear or cyclic.

Thus, included particularly are retro-inverso oligopeptidic compounds of the oligopeptidic compounds of the invention (and more particularly retro-inverso peptides). Retro-inverso oligopeptidic compounds comprise D-amino acids in reverse (opposite) order to the parental or reference compound sequence. A retro-inverso analogue thus has reversed termini and reversed order of e.g. peptide bonds, while approximately maintaining the topology of the side chains as in the parental or reference sequence.

The oligopeptidic compound may include partial retro-inverso sequences, i.e. a domain or part of a domain may comprise a retro-inverso sequence.

By “oligopeptidic compound” is meant a compound which is composed of amino acids or equivalent subunits, which are linked together by peptide or equivalent bonds. Thus, the term “oligopeptidic compound” includes peptides and peptidomimetics.

By “equivalent subunit” is meant a subunit which is structurally and functionally similar to an amino acid. The backbone moiety of the subunit may differ from a standard amino acid, e.g. it may incorporate one or more nitrogen atoms instead of one or more carbon atoms. In preferred embodiments, the subunit comprises a standard amino acid backbone, i.e. the backbone of a standard or coded amino acid. In other words, preferably the subunit is an amino acid. However, the amino acid subunit may comprise a non-standard (non-coded) R-group.

By “peptidomimetic” is meant a compound which is functionally equivalent or similar to a peptide and which can adopt a three-dimensional structure similar to its peptide counterparts, but which is not solely composed of amino acids linked by peptide bonds. A preferred class of peptidomimetics are peptoids, i.e. N-substituted glycines. Peptoids are closely related to their natural peptide counterparts, but they differ chemically in that their side chains are appended to nitrogen atoms along the molecule's backbone, rather than to the α-carbons as they are in amino acids.

Peptidomimetics, particularly non-peptidic molecules may be generated through various processes, including conformational-based drug design, screening, focused library design and classical medicinal chemistry. Not only may oligomers of unnatural amino acids or other organic building blocks be used, but also carbohydrates, heterocyclic or macrocyclic compounds or any organic molecule that comprises structural elements and conformation that provides a molecular electrostatic surface that mimics the same properties of the 3-dimensional conformation of the peptide may be used by methods known in the art.

Thus the peptidomimetics may bear little or no resemblance to a peptide backbone. Peptidomimetics may comprise an entirely synthetic non-peptide form (e.g. based on a carbohydrate backbone with appropriate substituents) or may retain one or more elements of the peptide on which it is based, e.g. by derivatizing one or more amino acids or replacing one or more amino acids with alternative non-peptide components. Peptide-like templates include pseudopeptides and cyclic peptides. Structural elements considered redundant for the function of the peptide may be minimized to retain a scaffold function only or removed where appropriate.

In preferred embodiments, peptidomimetics retain one or more peptide elements, i.e. more than one amino acid, although such amino acids may be replaced with a non-standard or structural analogue thereof. Amino acids retained in the sequences may also be derivatised or modified (e.g. labelled, glycosylated or methylated) as long as the functional properties of the oligopeptidic compound are retained. The peptidomimetics are referred to as being “derivable from” a certain polypeptide sequence. By this it is meant that the peptidomimetic is designed with reference to the peptide sequence defined above, such that it retains the structural features of the peptide which are essential for its function. This may be the particular side chains of the peptide, or hydrogen bonding potential of the structure. Such features may be provided by non-peptide components or one or more of the amino acid residues or the bonds linking said amino acid residues of the polypeptide may be modified so as to improve certain functions of the peptide such as stability or protease resistance, while retaining the structural features of the peptide which are essential for its function.

Examples of non-standard or structural analogue amino acids which may be used are D amino acids, amide isosteres (such as N-methyl amide, retro-inverse amide, thioamide, thioester, phosphonate, ketomethylene, hydroxymethylene, fluorovinyl, (E)-vinyl, methyleneamino, methylenethio or alkane), L-N methylamino acids, D-α methylamino acids, D-N-methylamino acids. Examples of non-conventional, i.e. non-coded, amino acids are listed in Table 2.

TABLE 2 Non-conventional Non-conventional amino acid Code amino acid Code α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine Nmorn D-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine Dile L-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophan Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrate Mgabu D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen D-α-methylasparagine Dmasn α-methyl-α-naphthylalanine Manap D-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-naphthylalanine Anap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycine Ncbut D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec D-α-methylvaline Dmval N-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg D-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine Nthr D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen N-methylglycine Nala D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nval D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine Pen L-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine Marg L-α-methylasparagine Masn L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug L-α-methylcysteine Mcys L-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamate Mglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet L-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine Mmet L-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithine Morn L-α-methylphenylalanine Mphe L-α-methylproline Mpro L-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl)carbamylmethyl)glycine Nnbhm N-(N-(3,3-diphenylpropyl)carbamylmethyl)glycine Nnbhe 1-carboxy-1-(2,2-diphenyl- Nmbc L-O-methyl serine Omser ethylamino)cyclopropane L-O-methyl homoserine Omhse

In preferred embodiments, the oligopeptidic compound is a peptide. In particularly preferred embodiments, the oligopeptidic compound is a peptide consisting of L-amino acids. In yet a further preferred embodiment, the oligopeptidic compound is a peptide consisting of standard or coded L-amino acids.

As mentioned above, the oligopeptidic compound may comprise non-standard amino acids. Thus, in some embodiments the oligopeptidic compound may incorporate di-amino acids and/or β-amino acids. However, in preferred embodiments, at least the APIM motif domain, consists of α-amino acids. Most preferably, the oligopeptidic compound, i.e. all domains and optionally all flanking sequences, consists of α-amino acids.

As mentioned above, the oligopeptidic compound defined herein comprises more than 5 subunits, but the length of the construct will depend on the size of the uptake peptide sequence and on the number and size of other domains, e.g. linker domains, flanking sequences etc, if present. Thus, the prefix “oligo” is used to designate a relatively small number of subunits such as amino acids, i.e. less than 200, preferably less than 150, 100, 90, 80, 70, 60 or 50 subunits. The oligopeptidic compound of the invention may thus comprise more than 5 but no more than 200 subunits. Preferably, it comprises at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 subunits. Alternatively defined it comprises no more than 50, 45, 40, 35, 34, 33, 32, 31 or 30 subunits. Representative subunit ranges thus include 12-50, 12-45, 12-40, 12-35, 12-30, 12-25, 12-22, 12-20, 12-18 etc, 12-30 and 12-40 being preferred.

The nature of the subunits of the oligopeptidic compound outside of the APIM motif domain and the uptake peptide sequence is not critical, so the subunits outside of the motif may for example be those found in a native protein comprising the motif, such as hABH2, or they may be alanine residues or any other suitable residues.

Peptidomimetics typically have a longer half life within a patient's body, so they may be preferred in embodiments where a longer lasting effect is desired. This can help reduce the frequency at which the composition has to be re-administered. However, for bio-safety reasons a shorter half life may be preferred in other embodiments; in those embodiments peptides are preferred.

The oligopeptidic compound may form part of a larger unit, e.g. it may be fused to a polypeptide to form a recombinant fusion protein or attached to a scaffold to form a peptide aptamer. Thus, fusion proteins or aptamers incorporating the oligopeptidic compound may also find utility in the uses and methods of the invention, i.e. in some embodiments the agent may be a fusion protein or aptamer comprising the oligopeptidic compound defined above.

Yet further aspects include pharmaceutical compositions comprising the agent defined herein, e.g. comprising the oligopeptidic compound, fusion protein or aptamer, together with at least one pharmacologically acceptable carrier or excipient, wherein said composition is for use in the uses and methods of the invention defined below.

In a further aspect, a nucleic acid molecule encoding a peptide having or comprising (e.g. of) SEQ ID NO: 1, as defined above, is provided for use in the methods and uses of the invention. Alternatively viewed, the agent for use in the uses and methods of the invention may be a nucleic acid molecule encoding a peptide having or comprising (e.g. of) SEQ ID NO: 1, as defined above. In this respect, the nucleic acid molecule may not need to encode all of the domains of the oligopeptidic compound described above, e.g. the domain that facilitates the cellular uptake of the peptide. For instance, the nucleic acid molecule may be delivered into the cell by another mechanism, e.g. via a liposome. However, in a preferred embodiment, the invention provides a nucleic acid molecule encoding an oligopeptidic compound or construct (e.g. a peptide) as defined above, comprising a PCNA interacting motif (APIM motif) domain and a peptide sequence (domain) that facilitates the uptake of said peptide. Also provided is the complement of such a nucleic acid molecule for use in the uses and methods of the invention. Thus, in some embodiments the nucleic acid molecule may also encode one or more linker and/or signal sequences, as defined above.

The nucleic acid molecule of the invention comprises at least 15 nucleotides, preferably at least 36 nucleotides, and preferably no more than 800 nucleotides, more preferably no more than 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 75 or 50 nucleotides. The nucleic acid molecule is preferably an isolated or synthetic molecule.

A further aspect relates to a vector comprising a nucleic acid molecule as defined herein for use in the uses and methods defined below. Preferably, the vector comprises a promoter sequence operably linked to the sequence encoding a peptide as defined above. The vector may also contain further elements typically found in a vector such as an origin of replication, a selectable marker such as antibiotic resistance, and/or a multiple cloning site. The vector may further be an expression vector, and may comprise further elements, e.g. transcriptional and/or translational control or regulatory elements for expression of the nucleic acid molecules. Such control elements, e.g. promoters, ribosome binding sites, enhancers, terminators etc. are well known and widely described in the art.

The vector may for example be a plasmid or a viral genome (or part thereof), preferably the viral gemone is from a virus selected from a retrovirus, an adenovirus and an adenovirus-associated virus. Thus, in some embodiments, the vector may be administered in the form of a virus comprising a vector containing a nucleic acid molecule encoding an oligopeptidic compound described above. Alternatively viewed, in some embodiments the vector may be a virus.

As mentioned above, there is provided a composition (e.g. a pharmaceutical composition) comprising an agent as defined herein for use in the methods and uses of the invention. Accordingly, said composition (e.g. a pharmaceutical composition) may comprise an oligopeptidic compound (including a fusion protein or aptamer) and/or nucleic acid molecule as defined herein and/or a vector as defined herein, together with at least one pharmacologically (or pharmaceutically) acceptable carrier or excipient.

The excipient may include any excipients known in the art, for example any carrier or diluent or any other ingredient or agent such as buffer, antioxidant, chelator, binder, coating, disintegrant, filler, flavour, colour, glidant, lubricant, preservative, sorbent and/or sweetener etc.

The excipient may be selected from, for example, lactic acid, dextrose, sodium metabisulfate, benzyl alcohol, polyethylene glycol, propylene glycol, microcrystalline cellulose, lactose, starch, chitosan, pregelatinized starch, calcium carbonate, calcium sulfate, dextrates, dextrin, dextrose, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, magnesium carbonate, magnesium oxide, maltodextrin, mannitol, powdered cellulose, sodium chloride, sorbitol and/or talc.

The pharmaceutical composition may be provided in any form known in the art, for example as a tablet, capsule, coated tablet, liquid, suspension, tab, sachet, implant, inhalant, powder, pellet, emulsion, lyophilisate, effervescent, spray, salve, emulsion, balm, plaster or any mixtures thereof. It may be provided e.g. as a gastric fluid-resistant preparation and/or in sustained action form. It may be a form suitable for oral, parenteral, topical, rectal, genital, subcutaneous, transurethral, transdermal, intranasal, intraperitoneal, intramuscular and/or intravenous administration and/or for administration by inhalation.

In a representative embodiment, the pharmaceutical composition may be in a form suitable for liposomal administration, so preferably liposomes containing the pharmaceutical composition are provided. When liposomes are used, it may not be necessary to include a further excipient, so also provided are liposomes containing an agent, e.g. oligopeptidic compound, as defined herein, for use in the methods and uses of the invention.

As mentioned above, the agents defined herein, i.e. oligopeptidic compounds and nucleic acid molecules, and pharmaceutical compositions comprising said agents exhibit therapeutic properties in the treatment of various conditions or disorders, particularly disorders associated with cytokine release from non-proliferating immune cells in blood and infectious diseases or disorders or conditions exacerbated or caused by an infection.

As referred to herein a “disorder”, “disease” or “condition” refers to an underlying pathological disturbance in a symptomatic or asymptomatic organism (subject) relative to a normal organism (subject), which may result, for example, from infection and/or an acquired or congenital genetic imperfection.

As set forth in the Examples, the inventors have determined that oligopeptidic compounds comprising a PCNA interacting motif can reduce cytokine release in blood, particularly from non-proliferating immune cells in the blood, e.g. monocytes. Cytokines may be released into blood on stimulation with a variety of substances and artificial/synthetic substances. For instance, LPS and poly I:C can be used to simulate or represent bacterial and viral infections, respectively. Excessive levels of pro-inflammatory cytokines are associated with a variety of diseases, e.g. infectious diseases, and it is expected that the agents defined herein may be effective in the treatment or prevention of said disorders. For example, the symptoms and complications associated with sepsis are thought to arise from the overproduction of pro-inflammatory cytokines in blood. The introduction or administration of the agents of the invention, e.g. intravenously, would therefore be expected to treat or prevent sepsis by reducing or inhibiting the release of said cytokines in blood. Thus, the agents described herein may be expected to find utility in the treatment or prevention of any disease or disorder associated with uncontrolled, undesirable or excessive cytokine release (e.g. hypercytokinemia) from immune cells in blood, e.g. particularly uncontrolled, undesirable or excessive release of one or more pro-inflammatory cytokines.

The inventors have demonstrated that some of the proteins involved in the toll-like receptor signal transduction pathways, which as discussed above are involved in the regulation of immune responses, comprise a PCNA interacting motif. Accordingly, it is thought that the oligopeptidic compounds disclosed herein are able to interfere with the interaction of said proteins with PCNA, e.g. in non-proliferating cells in blood, thereby inhibiting the release of pro-inflammatory cytokines into blood. Accordingly, the oligopeptidic compounds disclosed herein find particular utility in the treatment or prevention of a condition or disorder associated with cytokine release from non-proliferating immune cells in blood, particularly conditions or disorders associated with excessive, undesirable or uncontrolled pro-inflammatory cytokine release from non-proliferating immune cells in blood, e.g. an infectious disease or infection or a disease or condition exacerbated or caused by an infection.

In other words, the oligopeptidic compounds disclosed herein find particular utility in the treatment or prevention of excessive, undesirable or uncontrolled pro-inflammatory cytokine from non-proliferating immune cells in blood in a subject, e.g. a subject with an infectious disease or infection or a disease or condition exacerbated or caused by an infection.

Whilst not wishing to be bound by theory, it is thought that the effect of APIM peptides on cytokine release may be dependent on the strength of the interaction between the APIM peptide and its polypeptide target(s). Thus, as different APIM peptides may interact with target polypeptides with different affinities, it is thought that the dose of any one APIM peptide may determined independently. However, the dose for any APIM peptide may be determined as a matter of routine. In general, a suitable dose may be defined as a dose that does not induce apoptosis, i.e. an apoptosis non-inducing dose, which may be deduced by analysing the minimum concentration of oligopeptidic compound required to induce apopotosis in a cell, e.g. using a standard in vitro assay.

Since the therapeutic applications and utilities of the present invention may generally involve inhibiting the release of cytokines from non-proliferating immune cells in blood, any non-proliferating immune cells in blood may be targeted in the therapies and utilities disclosed and encompassed herein. Such non-proliferating immune cells may include healthy or diseased cells, and particularly cells that are involved in cytokine release. For example, such cells may include in particular monocytes, e.g. cells that display the cell surface receptor CD14 and/or the cell surface receptor CD16.

The cells to be targeted in the therapies and utilities disclosed and encompassed herein are particularly associated with the release of pro-inflammatory cytokines and chemokines. Thus, in some embodiments, in addition, or as an alternative, to the cell surface receptors displayed, the non-proliferating immune cells, e.g. monocytes, may be characterised or defined by their capacity to release (e.g. secrete) any one or more of the cytokines or chemokines selected from TNFα, IL1RA, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, CCL11, BasicFGF, G-CSF, GM-CSF, INFγ, CXCL10, CCL2, CCL3, CCL4, PDGF-β, CCL5 and VEGF.

Thus, in some embodiments, the non-proliferating immune cells are monocytes, particularly monocytes that display the cell surface receptor CD14 and/or the cell surface receptor CD16. In some embodiments the non-proliferating immune cells are monocytes that are capable of releasing (e.g. monocytes that secrete or release) any one or more of the cytokines or chemokines selected from TNFα, IL1RA, IL-β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, CCL11, BasicFGF, G-CSF, GM-CSF, INFγ, CXCL10, CCL2, CCL3, CCL4, PDGF-β, CCL5 and VEGF.

The term “treatment” as used herein refers broadly to any effect or step (or intervention) beneficial in the management of a clinical condition or disorder and thus includes both therapeutic and prophylactic treatments. Treatment may include reducing, alleviating, ameliorating, slowing the development of, or eliminating the condition or one or more symptoms thereof, which is being treated, relative to the condition or symptom prior to the treatment, or in any way improving the clinical status of the subject. A treatment may include any clinical step or intervention which contributes to, or is a part of, a treatment programme or regimen. A prophylactic treatment may include delaying, limiting, reducing or preventing the condition or the onset of the condition, or one or more symptoms thereof, for example relative to the condition or symptom prior to the prophylactic treatment. Prophylaxis thus explicitly includes both absolute prevention of occurrence or development of the condition, or symptom thereof, and any delay in the onset or development of the condition or symptom, or reduction or limitation on the development or progression of the condition or symptom.

Treatment according to some aspects of the invention thus includes reducing or lowering the levels or amounts of one or more cytokines released into the blood from non-proliferating immune cells, e.g. in response to an infection, such as a microbial, viral or parasitic infection. Treatment also includes the prevention or inhibition of the release into the blood of one or more cytokines from non-proliferating immune cells, e.g. the prevention or inhibition of the release of cytokines above a level or amount considered to be within a normal range. Thus, in some aspects of the invention, treatment may be viewed as maintaining the level or amount of one or more cytokines in blood within a normal range. A normal range may depend on the subject being treated. Thus, for instance, a normal range may be the amount or level of said one or more cytokines in a healthy subject. Alternatively, a normal range may be the average or typical level or amount of said one or more cytokines in subjects suffering from a particular disease or condition as defined herein, e.g. an infection. Hence, the treatment may involve reducing or lowering an excessive, unwanted or above normal level or amount of one or more cytokines that have been released into blood from non-proliferating immune cells. Treatment may also encompass the prevention or inhibition of the release of unwanted, excessive or above normal amounts or levels of one of more cytokines from non-proliferating immune cells in blood. The term “treatment” does not necessarily imply cure or complete abolition or elimination of unwanted, excessive or above normal cytokine release from non-proliferating immune cells in blood.

The term “disorder or condition associated with cytokine release from non-proliferating immune cells in blood” is used broadly herein to include any disorder or condition which involves increased, undesired, unwanted or excessive amounts or levels of cytokines in blood as a result of the release of cytokines from non-proliferating immune cells. Thus included are not only conditions in which the levels of one or more cytokines is increased in blood, for example relative to normal or healthy cytokine levels, or cytokine levels in the absence of the condition in question (e.g. compared or relative to a healthy or control subject, or compared or relative to cytokine levels taken from healthy or unaffected tissue in the same subject), but also conditions in which level of cytokines is not increased (or not greatly or significantly increased) over normal, but in which the cytokine level that occurs is unwanted or undesired, whether generally or in a particular context. This may include for example an unwanted or undesired release of cytokines which may occur in a “normal” response, e.g. an immune response or an inflammatory response etc. (in other words a “normal” response which may occur in a particular (e.g. normal) context, but which may nonetheless be unwanted). Such an unwanted cytokine release response may for example be the release of cytokines resulting in an unwanted inflammatory response, or an unwanted immune response such as an autoimmune response, an allergic reaction, rejection of a transplanted organ or tissue etc.

In some embodiments the disorder or condition associated with cytokine release from non-proliferating immune cells in blood may be characterised as hypercytokinemia, which may be viewed as an unwanted or excessive release, or increase in the level or amount, of one or more cytokines in blood. In some embodiments, hypercytokinemia may be a global cytokine release, comprising the unwanted (e.g. uncontrolled, excessive or above normal) release or increase of multiple cytokines, e.g. an increase in the level of at least 2 cytokines defined herein, such as an increase of at least 3, 4, 5, 6, 7, 8, 9, 10 or more cytokines as defined herein. A release or increase in multiple cytokines may be termed a “cytokine storm”. Accordingly, the disorder or condition to be treated may be a condition that results in, e.g. can lead to, hypercytokinemia, such as a cytokine storm, e.g. sepsis or malaria. In other embodiments, hypercytokinemia may be associated with the release of specific cytokines in blood, e.g. an increase in a particular cytokine. For instance, the excessive or increased release of interferon-β (INF-β) in response to a viral infection, e.g. an acute or persistent (chronic) virus infection, can ultimately lead to a number of inflammatory or autoimmune diseases, such as systemic lupus erythematosus, anaemia and other autoimmune diseases.

Thus, in some embodiments, the agent disclosed herein may be used as a preventative, prophylactic or protective agent against an unwanted or excessive (above normal) cytokine release (i.e. hypercytokinemia) or to maintain cytokine levels or amounts within a normal range, e.g. may be useful in treatment via the prevention of disorder or condition (or the exacerbation of a disorder or condition) associated with cytokine release from non-proliferating immune cells in blood. In some embodiments, the agent described herein may be used as a direct therapeutic agent, e.g. to treat a disorder or condition associated with hypercytokinemia, i.e. to facilitate the reduction in the levels or amounts of said unwanted or excessive (above normal) one or more cytokines in a subject in which a disorder or condition has already progressed to hypercytokinemia. Thus, in some embodiments the disorder or condition associated with cytokine release from non-proliferating immune cells in blood may be viewed as a disorder or condition that results in (may lead to) hypercytokinemia or a disorder or condition that is associated with hypercytokinemia. Alternatively viewed, the disorder or condition associated with cytokine release from non-proliferating immune cells in blood may be viewed as hypercytokinemia resulting from an associated disorder or condition, e.g. hypercytokinemia resulting from an infection.

An unwanted or excessive release (e.g. uncontrolled or above normal) or increase in the level or amount of one or more cytokines in blood may be defined as an increase in one or more cytokines by at least 10% relative to normal levels. As mentioned above, normal levels may be defined, e.g. relative to the levels in the blood of a healthy subject or the levels in another tissue of the subject to be treated. In some embodiments the normal levels may be defined relative to the average or typical levels in a subject with the disorder or condition to be treated or prevented, e.g. the average or typical levels in a subject at an early stage of the disorder or condition, i.e. a condition where progression of the condition will result in an undesirable release of one or more cytokines. In some embodiments, the unwanted or excessive release or increase may be an increase by at least 15, 20, 25, 30, 35, 40, 45 or 50%, e.g. at least 60, 70, 80, 90 or 100%. In still further embodiments, it may be an increase of at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 fold of normal levels, such as at least 15, 20, 30, 40, 50 or 100 fold normal levels. It will be evident that the range of the increase will, to some extent, be dependent on the nature of the one or more cytokines in question. Hence, the above values may be applied to a single cytokine or the increase may be measured as an average of the total increase, i.e. where more than one cytokine is increased.

A reduction of unwanted or excessive (e.g. uncontrolled or above normal) levels or amounts of one or more cytokines in blood may be defined as a decrease in one or more cytokines by at least 10%, e.g. relative to the levels prior to treatment. In some embodiments, the unwanted or excessive levels or amounts may be decreased by at least 15, 20, 25, 30, 35, 40, 45 or 50%, e.g. at least 60, 70, 80, 90 or 100%. In still further embodiments, it may be a decrease of at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 fold of the amounts or levels prior to treatment, such as at least 15, 20, 30, 40, 50 or 100 fold decrease. In some embodiments, the decrease may be a reduction of the amount or level to within about 25% or less of the normal level, wherein the normal level is as defined above. Thus, the decrease may be a reduction to within about 20, 15, 10, 5% or less of the normal level. It will be evident that the range of the decrease or reduction will, to some extent, be dependent on the nature of the one or more cytokines in question. Hence, the above values may be applied to a single cytokine or the decrease or reduction may be measured as an average of the total decrease, i.e. where the level or amount of more than one cytokine is decreased.

In some embodiments, the agent or composition results in a reduction of cytokines and/or chemokines selected from any one or more of TNFα, IL1RA, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, CCL11, BasicFGF, G-CSF, GM-CSF, INFγ, CXCL10, CCL2, CCL3, CCL4, PDGF-β, CCL5 and VEGF. In some embodiments, the agent or composition results in a reduction of one or more of TNFα, IL1RA, IL-1β, IL-2, IL-4, IL-6, IL-8 (CXCL8), IL-9, IL-10, IL-17, G-CSF, INFγ, CXCL10, CCL2, CCL3 and CCL4 (e.g. in a disease or condition associated with a bacterial infection). In further embodiments, the agent or composition results in a reduction of one or more of TNFα, IL1RA, IL-1β, IL-2, IL-4, IL-6, IL-8 (CXCL8), IL-9, IL-17, G-CSF and INFγ (e.g. in a disease or condition associated with a bacterial infection). In some more particular embodiments, the agent or composition results in a reduction of one or more of IFNγ, IL-1RA, IL-2, 4, and 9 (e.g. in a disease or condition associated with a bacterial infection). In yet further embodiments, the agent or composition results in a reduction of one or more of TNFα, IL-1β, IL-8 (CXCL8), CXCL10, CCL2, CCL3 and CCL4 (e.g. in a disease or condition associated with a viral infection). In some embodiments, the agent or composition results in a reduction of one or more of TNFα, IL-8 (CXCL8), CXCL10, CCL2, CCL3 and CCL4 (e.g. in a disease or condition associated with a viral infection). In some more particular embodiments the agent or composition results in a reduction of one or more of IL-1RA, CCL2, 3, and 4 (e.g. in a disease or condition associated with a viral infection).

The identification, characterisation, diagnosis and/or progression of the disorder or condition to be treated and/or the efficacy of the treatment may be facilitated by the measurement of one or more cytokines in the blood. Various assays for measuring cytokines are well known in the art and standard kits are commercially available. Typical methods rely on immunoassays, e.g. ELISAs, RIAs, etc., and may be performed using a variety of blood samples, such as serum, plasma or whole blood.

Thus, in a general embodiment the invention the disorder or condition associated with cytokine release from non-proliferating immune cells in blood is an inflammatory disease (e.g. inflammatory arthritis, psoriatic arthritis, rheumatoid arthritis, inflammatory bowel disease) or an autoimmune disease (e.g. systemic lupus erythematosus). Accordingly, the invention may be seen to provide an agent or composition as defined herein for use in the treatment or prevention of an inflammatory or autoimmune disease or a method for treating or preventing an inflammatory or autoimmune disease comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof. Alternatively viewed, the invention may be seen to provide an agent or composition as defined herein for use in the treatment or prevention of hypercytokinemia in a subject with an inflammatory or autoimmune disease or a method of treating or preventing hypercytokinemia in a subject with an inflammatory or autoimmune disease comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof.

In some more particular embodiments of the invention, the disorder or condition associated with cytokine release from non-proliferating immune cells in blood is an infection or infectious disease, i.e. the agent described herein may be for use in treating an infection or infectious disease or the invention may be seen to provide a method of treating a subject with an infection or infectious disease comprising administering (particularly administering an effective amount of) an agent as described herein to a subject in need thereof. Alternatively viewed, the invention may provide an agent or composition as described herein for use in the treatment or prevention of hypercytokinemia in a subject with an infection or infectious disease or a method of treating or preventing hypercytokinemia in a subject with an infection or infectious disease comprising administering (particularly administering an effective amount of) an agent or composition as described herein to a subject in need thereof. The invention also extends to the use of the agent in the manufacture of a medicament for the above utilities.

An “infection” or “infectious disease” may be defined as a disease, condition or disorder caused by the invasion of a subject, e.g. one or more organs or tissues of said subject, by one or more disease-causing organisms and their subsequent multiplication. In some instances, an infection or infectious disease may be characterised by the reaction of the subject (e.g. organ or tissues of said subject) to said organisms and, in some cases, to the toxins produced by said organisms. An infection or infectious disease may be a microbial, viral or parasitic infection and may be local or systemic. A microbial infection may be any bacterial or fungal infection, i.e. caused by a bacterium or fungus. In particularly preferred embodiments, the disease, condition or disorder caused by a viral infection is not a viral-induced hyperproliferative disease, such as warts and EBV-induced disease (e.g. infectious mononucleosis), scar formation and the like.

In some embodiments, the infectious disease or infection may be a bacterial infection or disease. Examples of bacteria that cause infections or infectious diseases that may be treated or prevented by the agents or compositions described herein may be gram positive or gram negative, or gram test non-responsive. They may be aerobic or anaerobic bacteria. For instance, the bacteria may be from any of the genus Achromobacter, Acinetobacter, Actinobacillus, Aeromonas, Agrobacterium, Alcaligenes, Alteromonas, Bacillus, Bacteroides, Bartonella, Borrelia, Bordetella, Brucella, Burkholderia, Campylobacter, Cardiobacterium, Chlamydia, Chlamydophila, Chromobacterium, Chyseobacterium, Chryseomonas, Citrobacter, Clostridium, Comamonas, Corynebacterium, Coxiella, Cryptobacterium, Edwardsiella, Eikenefia, Enterobacter, Enterococcus, Erwinia, Helicobacter, Kingella, Klebsiella, Lactobacillus, Lactococcus, Legionella, Leptospira, Leptotrichia, Leuconostoc, Listeria, Listonella, Mobiluncus, Moraxella, Morganella, Mycobacterium, Mycoplasma, Neisseria, Nocardia, Nocardiopsis, Pantoea, Parachlamydia, Pasteurella, Peptococcus, Peptostreptococcus, Prevotella, Propionibacterium, Proteus, Providencia, Pseudomonas, Ralstonia, Rickettsia, Salmonella, Shewenella, Shigella, Sphingobacterium, Sphingomonas, Staphylococcus, Stenotrophomonas, Streptobacillus, Streptococcus, Streptomyces, Treponem and Yersinia, such as Acinetobacter, Bacillus, Burkholderia, Chlamydia, Clostridium, Helicobacter, Staphylococcus, Streptococcus, Pseudomonas, Legionella, Listeria, Mycobacterium, Proteus, Klebsiella, Fusobacterium or other enteric or coliform bacteria.

Thus, for instance, the infection or infectious disease may be caused by a gram-positive bacteria such as, M. tuberculosis, M. bovis, M. typhimurium, M. bovis strain BCG, BCG substrains, M. avium, M. intracellulare, M. africanum, M. kansasii, M. marinum, M. ulcerans, M. avium subspecies paratuberculosis, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus equi, Streptococcus pyogenes, Streptococcus agalactiae, Listeria monocytogenes, Listeria ivanovii, Bacillus anthracis, B. subtilis, Nocardia asteroides, Actinomyces israelii, Propionibacterium acnes, and Enterococcus species.

In other embodiments, the infection or infectious disease may be caused by a gram-negative bacteria such as Clostridium tetani, Clostridium perfringens, Clostridium botulinum, Pseudomonas aeruginosa, Vibrio cholerae, Actinobacillus pleuropneumoniae, Pasteurella haemolytica, Pasteurella multocida, Legionella pneumophila, Salmonella typhi, Brucella abortus, Chlamydi trachomatis, Chlamydia psittaci, Coxiella bumetti, Escherichia coli, Neiserria meningitidis, Neiserria gonorrhea, Haemophilus influenzae, Haemophilus ducreyi, Yersinia pestis, Yersinia enterolitica, Escherichia coli, E. hirae, Burkholderia cepacia, Burkholderia pseudomallei, Francisella tularensis, Bacteroides fragilis, Fusobascterium nucleatum, and Cowdria ruminantium.

Thus, a disorder or condition associated with cytokine release from non-proliferating immune cells in blood may be the result of, e.g. caused by, a bacterial infection or infectious disease, such as sepsis.

In some embodiments, a bacterial infection may be associated with a particular disorder or condition, i.e. subjects suffering from a particular disorder or condition may be particularly susceptible to one or more bacterial infections. For instance, it is common for subjects with cystic fibrosis to suffer from chronic Pseudomonas infections. Thus, invention may be seen to extend to an agent or composition as defined herein for use in treating a disease or condition (e.g. hypercytokinemia) exacerbated or caused by an infection, e.g. bacterial pneumonia, cystic fibrosis, gastric ulcers, bacterial meningitis, Legionellosis (Legionnaires' disease), Legionellosis (Pontiac fever), Pertussis (Whooping cough), Salmonellosis, Tuberculosis etc.

Thus, in some embodiments a condition or disease caused by an infection is sepsis, which is a condition that results from a severe infection. Accordingly, the invention may be seen to provide an agent or composition as defined herein for use in the treatment or prevention of sepsis or a method for treating or preventing sepsis comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof. Alternatively viewed, the invention may be seen to provide an agent or composition as defined herein for use in the treatment or prevention of hypercytokinemia in a subject with sepsis or a method of treating or preventing hypercytokinemia in a subject with sepsis comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof.

In some embodiments a condition or disease exacerbated by an infection is cystic fibrosis, which is a condition that is commonly associated with a chronic Pseudomonas infection. Accordingly, the invention may be seen to provide an agent or composition as defined herein for use in the treatment of cystic fibrosis (or more particularly, treatment of a subject with cystic fibrosis, e.g. suffering from an infection, i.e. alleviating the one or more symptoms of a subject suffering from cystic fibrosis). Thus, the invention provides a method for treating cystic fibrosis (or more particularly, treating a subject with cystic fibrosis, e.g. suffering from an infection, i.e. alleviating the one or more symptoms of a subject suffering from cystic fibrosis) comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof.

In some embodiments, the infectious disease or infection may be a fungal infection or disease. In some aspects of the invention the fungus may be a mold or yeast, preferably a yeast. The fungus may be selected from any one or more of a Dermatophyte, Aspergillus sp. (such as Aspergillus fumigatus, Aspergillus nigricans or flavescens), Zygomycota sp., Fusarium sp., Trichophyton sp, Basidiobolus ranarum, Piedraia sp. (such as Piedraia hortae), Blastomyces dermatitidis, Candida sp. (such as Candida albicans), Chrysosporium, Coccidioides sp. (such as Coccidioides immitis and Coccidioides posadasii), Conidiobolus sp. (such as Conidiobolus coronatus and Conidiobolus incongruus), Cryptococcus sp. (such as Cryptococcus gattii and Cryptococcus neoformans), Histoplasma sp. (such as Histoplasma farciminosum and Histoplasma capsulatum), Exserohilum rostratum, Cladosporium sp., Saccharomyces sp., Lacazia loboi, Paracoccidioides brasiliensis, Penicillium mameffei, Pneumocystis jirovecii, Sporothrix schenckii, Diheterospora zeaspora, Absidia corymbifera, Apophysomyces elegans, Mucor indicus, Rhizomucor pusillus, Rhizopus oryzae, Cunninghamella bertholletiae, Cokeromyces recurvatus, Saksenaea vasiformis, Syncephalastrum racemosum, and Conidiobolus sp. (such as Conidiobolus coronatus and Conidiobolus incongruus).

By way of a representative example, fungi that cause infections or infectious diseases that may be treated or prevented by the agents and compositions described herein include fungi from the genera Candida, Aspergillus, Pneumocystis, Penicillium and Fusarium. Representative fungal species include, but are not limited to, Candida albicans, Candida dubliniensis, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidioides brasiliensis, Blastomyces dermitidis, Pneomocystis carnii, Penicillium marneffi, and Alternaria alternate. etc.

A variety of environmental and physiological conditions can contribute to the development of fungal diseases and infections. Fungal infections (mycoses) commonly start in the lungs or on the skin, e.g. a fungal infection (a mycosis) may result from the inhalation of fungal spores or localized colonization of the skin may initiate persistent infections. Thus, a disease or condition exacerbated or caused by a fungal infection (mycosis) to be treated or prevented by the agent or composition of the invention may be in any tissue or organ of the subject to be treated, such as the lungs (including the respiratory tract), skin (including wounds), mouth, ear, eye etc. Hence, the fungal infection may be a respiratory infection, skin infection, ear infection, eye infection etc.

Thus, a disorder or condition associated with cytokine release from non-proliferating immune cells in blood may be the result of, e.g. caused by, a fungal infection or infectious disease, caused by any one or more of the fungi (or fungi from one or more the genera) described above.

Thus, more generally, a disorder or condition associated with cytokine release from non-proliferating immune cells in blood may be the result of, or exacerbated by, a microbial infection, such as a microbe selected from any one or more of the genera Actinebacter, Citrobacter, Enterobacter, Escherichia, Hafnia, Serratia, Yersinia, Peptostreptococcus, Bacteroides, Pseudomonas, Legionella, Staphylococcus, Enterococcus, Streptococcus, Klebsiella, Candida, Proteus, Burkholderia, Fusobacterium and Mycobacterium, for instance, Staphylococcus aureus, Staphylococcus epidermidis, Legionella pneumophila, Candida albicans, Pseudomonas aeruginosa, Burkholderia cepacia and Streptococcus Pyogenes.

In some embodiments, the infectious disease or infection may be a viral infection or disease. Examples of viruses that cause infections or infectious diseases that may be treated or prevented by the agents described herein include Australian bat lyssavirus, Banna virus, Barmah forest virus, Bunyamwera virus, Bunyavirus La Crosse, Bunyavirus snowshoe hare, Cercopithecine herpesvirus, Chikungunya virus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Dhori virus, Dugbe virus, Duvenhage virus, Eastern equine encephalitis virus, Ebolavirus, Echovirus, Encephalomyocarditis virus, European bat lyssavirus, Hantaan virus, Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Hepatitis delta virus, Human herpesvirus 1, Human adenovirus, Human astrovirus, Human coronavirus, Human cytomegalovirus, Human enterovirus 68-70, Human herpesvirus 2, Human herpesvirus 6, Human herpesvirus 7, Human herpesvirus 8, Human immunodeficiency virus, Human parainfluenza, Human parvovirus B19, Human respiratory syncytial virus, Human rhinovirus, Human SARS coronavirus, Human T-lymphotropic virus, Human torovirus, Influenza A virus, Influenza B virus, Influenza C virus, Isfahan virus, JC polyomavirus, Japanese encephalitis virus, Junin arenavirus, KI Polyomavirus, Kunjin virus, Lagos bat virus, Lake Victoria marburgvirus, Langat virus, Lassa virus, Lordsdale virus, Louping ill virus, Lymphocytic choriomeningitis virus, Machupo virus, Mayaro virus, Measles virus, Mengo encephalomyocarditis virus, Mokola virus, Molluscum contagiosum virus, Monkeypox virus, Mumps virus, Murray valley encephalitis virus, New York virus, Nipah virus, Norwalk virus, O′nyong-nyong virus, Orf virus, Oropouche virus, Pichinde virus, Poliovirus, Punta toro phlebovirus, Puumala virus, Rabies virus, Rift valley fever virus, Ross river virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus, Sandfly fever sicilian virus, Sapporo virus, Semliki forest virus, Seoul virus, Sindbis virus, Southampton virus, St. louis encephalitis virus, Tick-borne powassan virus, Toscana virus, Uukuniemi virus, Varicella-zoster virus, Variola virus, Venezuelan equine encephalitis virus, Vesicular stomatitis virus, Western equine encephalitis virus, West Nile virus and Yellow fever virus.

By way of a representative example, viruses that cause infections or infectious diseases that may be treated or prevented by the agents and compositions described herein may be particularly selected from any one or more of Dengue virus, Ebolavirus, Echovirus, Encephalomyocarditis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Hepatitis delta virus, Human herpesvirus 1, Human adenovirus, Human astrovirus, Human coronavirus, Human cytomegalovirus, Human enterovirus 68-70, Human herpesvirus 2, Human herpesvirus 6, Human herpesvirus 7, Human herpesvirus 8, Human immunodeficiency virus, Human parainfluenza, Human parvovirus B19, Human respiratory syncytial virus, Human rhinovirus, Human SARS coronavirus, Human T-lymphotropic virus, Human torovirus, Influenza A virus, Influenza B virus, Influenza C virus, Japanese encephalitis virus, Measles virus, Mumps virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, West Nile virus and Yellow fever virus.

However, as mentioned above, in particularly preferred embodiments, the viral infection is not an infection that causes or results in viral-induced hyperproliferative disease, such as warts and EBV-induced disease (e.g. infectious mononucleosis), scar formation and the like.

Thus, a disorder or condition associated with cytokine release from non-proliferating immune cells in blood may be the result of, e.g. caused by, a viral infection or infectious disease, such as AIDS/HIV, Dengue fever, measles, mumps, rubella, influenza and hepatitis.

Accordingly, the invention may be seen to provide an agent or composition as defined herein for use in the treatment of a viral infection, e.g. AIDS/HIV, Dengue fever, measles, mumps, rubella, influenza or hepatitis (or the treatment of the symptoms of a viral infection, e.g. AIDS/HIV, Dengue fever, measles, mumps, rubella, influenza or hepatitis) or a method for treating a viral infection, e.g. AIDS/HIV, Dengue fever, measles, mumps, rubella, influenza or hepatitis (or treating a subject suffering from a viral infection, e.g. AIDS/HIV, Dengue fever, measles, mumps, rubella, influenza or hepatitis) comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof. Alternatively viewed, the invention may be seen to provide an agent or composition as defined herein for use in the treatment or prevention of hypercytokinemia in a subject with a viral infection, e.g. AIDS/HIV, Dengue fever, measles, mumps, rubella, influenza or hepatitis, or a method of treating or preventing hypercytokinemia in a subject with a viral infection, e.g. AIDS/HIV, Dengue fever, measles, mumps, rubella, influenza or hepatitis, comprising administering (particularly administering an effective amount of) an agent as defined herein to a subject in need thereof.

In some embodiments, the infectious disease or infection may be a parasitic infection or disease. Examples of parasites that cause infections or infectious diseases that may be treated or prevented by the agents and compositions described herein include Plasmodium sp., such as Plasmodium falciparum, Plasmodium knowlesi, Plasmodium vivax, Plasmodium berghei and Plasmodium yoelii infections, protozoa such as Toxoplasma species e.g. Toxoplasma gondii, Trypanosoma brucei, Trypanosoma cruzi, Leishmania species such as Leishmania major, Schistosoma such as Schistosoma mansoni and Entamoeba histolytica.

Thus, a disorder or condition associated with cytokine release from non-proliferating immune cells in blood may be the result of, e.g. caused by, a parasitic infection or infectious disease, such as malaria, toxoplasmosis, trypanosomiasis and schistosomiasis.

In further embodiments a condition or disease caused by an infection is a parasitic infection, such as malaria, which is a condition that results from a parasitic infection, e.g. Plasmodium infection, such as Plasmodium falciparum, Plasmodium knowlesi, Plasmodium vivax, Plasmodium berghei or Plasmodium yoelii infection. Accordingly, the invention may be seen to provide an agent or composition as defined herein for use in the treatment of a parasitic infection, e.g. malaria, toxoplasmosis, trypanosomiasis or schistosomiasis, (or the treatment of the symptoms of a parasitic infection, e.g. malaria, toxoplasmosis, trypanosomiasis or schistosomiasis) or a method for treating a parasitic infection, e.g. malaria, toxoplasmosis, trypanosomiasis or schistosomiasis, (or treating a subject suffering from a parasitic infection, e.g. malaria, toxoplasmosis, trypanosomiasis or schistosomiasis) comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof. Alternatively viewed, the invention may be seen to provide an agent or composition as defined herein for use in the treatment or prevention of hypercytokinemia in a subject with a parasitic infection, e.g. malaria, toxoplasmosis, trypanosomiasis or schistosomiasis, or a method of treating or preventing hypercytokinemia in a subject with a parasitic infection, e.g. malaria, toxoplasmosis, trypanosomiasis or schistosomiasis, comprising administering (particularly administering an effective amount of) an agent as defined herein to a subject in need thereof.

In yet further embodiments, the disorder or condition associated with cytokine release from non-proliferating immune cells in blood is graft versus host disease (GVHD), i.e. an immune response associated with the transplantation of cells, tissues or organs to a recipient from a genetically non-identical donor of the same species. The transplant is may be viewed as an allograft, allogeneic transplant or homograft. In some embodiments of the invention the transplant may be a stem cell and/or bone marrow transplant. Accordingly, the agent or composition may be for use in treating or preventing graft versus host disease or the invention may be seen to provide a method of treating a subject with graft versus host disease comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof. Alternatively viewed, the invention may provide an agent or composition as defined herein for use in the treatment or prevention of hypercytokinemia in a subject with a graft versus host disease or a method of treating or preventing hypercytokinemia in a subject with a graft versus host disease comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof. The invention also extends to the use of the agent in the manufacture of a medicament for the above utilities.

In yet further embodiments, the disorder or condition associated with cytokine release from non-proliferating immune cells in blood is trauma, e.g. trauma caused by injury (i.e. a physical wound or injury, such as a fracture or blow, e.g. a polytrauma (affecting multiple sites), a head trauma, chest trauma, abdominal trauma or extremity trauma) or surgery. In some cases, the injury or wound resulting from trauma may be a burn. A burn may be caused by heat, electricity, chemicals, friction or radiation. In some embodiments, the burn is a partial-thickness or second-degree burn (comprising damage to some of the underlying skin layers). In some embodiments, the burn is a full-thickness or third-degree burn (comprising damage that extends to all layers of the skin). In some embodiments, the burn is a fourth-degree burn, which additionally involves injury to deeper tissues, such as muscle or bone.

Accordingly, the agent or composition may be for use in treating trauma (e.g. caused by injury or surgery) or the invention may be seen to provide a method of treating a subject with trauma (e.g. caused by injury or surgery) comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof. Alternatively viewed, the invention may provide an agent or composition as defined herein for use in the treatment or prevention of hypercytokinemia in a subject with trauma (e.g. caused by injury or surgery) or a method of treating or preventing hypercytokinemia in a subject with trauma (e.g. caused by injury or surgery) comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof. The invention also extends to the use of the agent in the manufacture of a medicament for the above utilities.

In some subjects hypercytokinemia, which may be a result of a disease or condition as described herein, such as sepsis, malaria, graft versus host disease (GVHD) etc. can result in, or increase the risk of, multiple organ dysfunction syndrome (MODS), which is also known as multiple organ failure (MOF) or multisystem organ failure (MSOF). Thus, in some embodiments the invention provides an agent or composition as defined herein for use in the treatment or prevention of MODS or a method for treating or preventing MODS comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof. Alternatively viewed, the invention may be seen to provide an agent or composition as defined herein for use in the treatment or prevention of hypercytokinemia in a subject with MODS or a method of treating or preventing hypercytokinemia in a subject with MODS comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof.

In some embodiments, the agent or composition as defined herein may be useful for the prevention of hypercytokinemia in a subject suffering from, or at risk from, any of the conditions mentioned above.

As noted above, in some embodiments the agent or composition as defined herein is used in combination with one or more additional active agents, e.g. an immunosuppressive compound, an anti-inflammatory compound, anti-microbial compound, or steroid (e.g. a corticosteroid) or a kinase inhibitor (such as a p38 MAPK inhibitor or a class I PI3K inhibitor), in order to enhance or complement the effect of the agent or composition defined herein, e.g. to treat symptoms of the disease or condition that are not directly affected by the agent or composition of the invention. However, in some embodiments, the agent as defined herein may be used alone, i.e. as the only active agent in a composition and/or medicament.

In some embodiments, the additional active agent is an immunosuppressive compound. Suitable immunosuppressive compounds include but are not limited to any one or more of glucocorticoids, cyclophosphamide, methotrexate, azathioprine, mercaptopurine, dactinomycin, anthracyclines, mitomycin C, bleomycin and mithramycin.

In some embodiments, the additional active agent is an anti-inflammatory compound. Suitable anti-inflammatory compounds include but are not limited to any one or more of diclofenac, ibuprofen, naproxen, celecoxib, mefenamic acid, etoricoxib, indometacin and aspirin.

In some embodiments, the additional active agent is an antibiotic compound. Suitable antibiotic compounds include but are not limited to any one or more of Aminocoumarins (such as Novobiocin, Albamycin, Coumermycin and Clorobiocin), Aminoglycosides (such as Amikacin, Apramycin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin and Spectinomycin), Ansamycins (such as Geldanamycin, Herbimycin, Rifaximin and Streptomycin), Carbapenems (such as Ertapenem, Doripenem, Cilastatin (‘Imipenem’) and Meropenem), Cephalosporins (such as Cefadroxil, Cefazolin, Cefalothin (‘Cefalotin’), Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil and Ceftobiprole) Glycopeptides (such as Teicoplanin, Vancomycin and Telavancin), Lincosamides (such as Clindamycin and Lincomycin), Lipopeptides (such as Daptomycin), Macrolides (such as Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin and Spiramycin), Monobactams (such as Aztreonam), Nitrofurans (such as Furazolidone and Nitrofurantoin), Oxazolidonones (such as Linezolid, Posizolid, Radezolid and Torezolid), Penicillins (such as Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Temocillin and Ticarcillin), Penicillin combinations (such as Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam and Ticarcillin/clavulanate), Polyethers (such as Monensin), Polypeptides (such as Bacitracin, Colistin and Polymyxin B), Quinolones (such as Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin and Temafloxacin); Sulfonamides (such as Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Sulfamethoxazole (Co-trimoxazole, TMP-SMX, ‘Trimethoprim’) and Sulfonamidochrysoidine), Tetracyclines (such as Demeclocycline, Doxycycline, Minocycline, Oxytetracycline and Tetracycline) and Others (such as Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin (‘Rifampin’), Rifabutin, Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid, Metronidazole, Mupirocin, Platensimycin, Quinupristin (Dalfopristin), Thiamphenicol, Tigecycline, Tinidazole and Trimethoprim).

In some embodiments, the additional active agent is an antifungal compound. Suitable antifungal compounds (antimycotics) include but are not limited to any one or more of Polyene antifungals (such as Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin and Rimocidin), Imidazoles (such as Bifonazole, Butoconazole, Clotrimazole, Econazole, Fenticonazole, Isoconazole, Ketoconazole, Miconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole and Tioconazole), Triazoles (such as Albaconazole, Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Ravuconazole, Terconazole and Voriconazole), Thiazoles (such as Abafungin), Allylamines (such as Amorolfin, Butenafine, Naftifine and Terbinafine), Echinocandins (such as Anidulafungin, Caspofungin and Micafungin) and Others such as Benzoic acid, Ciclopirox, Flucytosine or 5-fluorocytosine, Griseofulvin, Haloprogin, Polygodial, Tolnaftate, Undecylenic acid and Crystal violet.

In some embodiments, the additional active agent is a kinase inhibitor. Suitable kinase inhibitors include but are not limited to any one or more of p38 MAPK inhibitors (such as VX-702, SB203580, VX-745, LY2228820, BIRB 796 (Doramapimod), PH-797804 and TAK-715) and class I PI3K inhibitors (such as AS-605240, BYL719, CAL-101, GDC-0941 (Bismesylate salt), GDC-0941 (free base), GSK2636771, IC-87114, IPI-145, LY294002, NVP-BKM120 (Buparlisib), PIK-75, TG100-115 and TGX-221).

As discussed above, a suitable dose may be defined as a dose that does not induce apoptosis, i.e. an apoptosis non-inducing dose. In some embodiments, a suitable dose may be defined as a “low dose” or “low amount” of the agent (e.g. oligopeptidic compound), which may be seen as a dose or amount that is not sufficient to cause or induce apoptosis either directly or indirectly. Alternatively viewed, a “low dose” or “low amount” of the agent is a dose or amount that is an effective dose or amount for reducing or inhibiting cytokine release from non-proliferating immune cells in blood.

Accordingly, a high dose or amount may be viewed as an effective dose or amount that is sufficient to cause or induce apoptosis either directly or indirectly (i.e. cytotoxic doses or doses that result in an increased sensitivity to other cytotoxic or cytostatic agents).

The effective dose or amount of agent may depend on the characteristics of the peptide, e.g. the strength of the interaction between the PCNA interacting motif and the binding domain of the target protein(s). Furthermore, effective dose or amount of the agent may depend upon the nature of the compound used (i.e. peptide, nucleic acid molecule etc), the mode of administration, the course of treatment, the age and weight of the patient, the medical indication, the body or body area to be treated and may be varied or adjusted according to choice. Generally however, a low dose or amount may result in an active concentration range of about 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, 1.0, 1.25, 1.50, 1.75, 2.0, 3.0, 4.0, 5.0 to 10 μM, e.g. 0.01 to 10 μM, e.g. 0.05 to 7.5 μM, such as 0.1 to 7.5 μM, e.g. 0.5 to 5 μM. A high dose or amount may result in an active concentration range of about 1.0, 2.0, 3.0, 4.0, 5.0, 7.5, 10, 15, 20, 25, 30, 40 to 50 μM, e.g. 1.0 to 50 μM, e.g. 2.0 to 40 μM, such as 3.0 to 30 μM, e.g. 5.0 to 25 μM. Said concentrations are determined by reference to the amount of the compound itself and thus appropriate allowances should be made to take into account the purity of the composition.

The subject is an animal (i.e. any human or non-human animal), preferably a mammal, most preferably a human.

The skilled person will be well aware of suitable methods for introducing the agents as defined herein into cells. By way of example, a few suitable methods are briefly discussed below. As discussed in detail above, peptide-mediated methods of delivery can be used, notably uptake (import) peptides, such as cell penetrating peptides (CPPs), which as discussed above, are short, in some cases polycationic, sequences which can facilitate cellular uptake of peptides, proteins or nucleotide molecules which contain CPPs or to which CPPs are linked, for example by enhancing uptake into endosomes of mammalian cells. As mentioned above, the oligopeptidic compound of defined herein typically comprises an uptake peptide, e.g. a CPP. However, additional mechanisms to facilitate the uptake of the agents of the invention may be useful, particularly for agents comprising or consisting of nucleic acid molecules. Microencapsulation provides a simple and cost-effective way to enclose bioactive materials within a semi-permeable polymeric membrane for the purpose of protecting the bioactive materials and releasing the enclosed substances or their products in a controlled fashion. In photochemical internalisation (PCI) both the molecule of interest and a photosensitising compound are taken up by the cell into a lysosome or an endosome. The cells are then exposed to light of suitable wavelengths to activate the photosensitising compound, causing the photosensitising compound to disrupt the membrane of the lysosome or endosome, thereby releasing the molecule of interest into the cytosol of the cell.

Other methods include microinjection, red blood cell ghost-mediated fusion, liposome fusion, osmotic lysis of pinosomes, scrape loading, electroporation, calcium phosphate and virus-mediated transfection and the use of copolymeric carriers.

Chitosan and water-soluble chitosan derivatives, in particular glycol chitosan, are emerging as the drug carriers of choice because of their biocompatibility and biodegradability in vivo. A preferred example is glycol chitosan hydrophobically modified with 5β-cholanic acid.

The invention will now be further described with reference to the following non-limiting Examples and Figures in which:

FIG. 1 shows that ATX-101 (SEQ ID NO: 1198, which contains the APIM motif, RWLVK (SEQ ID NO: 28)) affects MAPK and PI3K/Akt pathways in multiple myeloma cell lines, wherein: (A) shows cell growth (MTT assay) of JJN-3 cells untreated (♦) and after addition of 6 μM ATX-101 (▴), 5 μM LY294002 (PI3K class I inhibitor) and 10 μM SB20358 (p38 MAPK inhibitor) (●) (left and right panel, respectively), and combination of ATX-101 and kinase inhibitor (□). Data is from one representative experiment out of three; and (B) shows quantification of p70 S6 kinase Thr389, Akt Ser473, ERK1 Thr202/Tyr204, and ERK2 Thr185/Tyr187 phosphorylation in JJN-3 cells after 4, 8, and 24 h treatment with 6 μM ATX-101 by Western blot analysis. Phosphorylated kinase levels were corrected for loading differences (β-tubulin) and normalized to untreated control cells. Data is from three independent experiments (mean±SD).

FIG. 2 shows that ATX-101 reduces cytokine secretion of monocytes after TLR stimulation, wherein: (A) and (B) show multiplex analysis of cytokine levels produced by peripheral blood monocytes after 4 h treatment with 4 μM ATX-101 in combination with 10 ng/ml LPS (A) and 40 μg/ml polyIC stimulation (B). Measured cytokine levels were normalized to cytokine levels from monocytes stimulated with TLR ligand alone. A two fold or higher increase in cytokine level after TLR stimulation was defined as cytokine induction. Data is from three donors (mean±SD); (C) shows an ELISA of IFN-β levels produced by peripheral blood monocytes from five donors. Isolated monocytes were stimulated with 10 ng/ml LPS and 40 μg/ml polyIC alone and in combination with 4 μM ATX-101 for 4 h. Donor 1 was treated with 6 μM ATX-101. # Under linear range;

over linear range; and (D) shows a confocal fluorescence image of immunofluorescently stained PCNA (green) in freshly isolated peripheral blood monocytes. DRAQ5 was used for nuclear staining (red). Bar, 5 μm.

FIG. 3 shows that ATX-101 reduces Akt phosphorylation in monocytes, wherein: (A) shows a Western blot analysis of Akt Ser473 phosphorylation in peripheral blood monocytes stimulated with 10 ng/ml LPS and 40 μg/ml polyIC alone and in combination with 4 μM ATX-101 for 4 h. The upper panel shows the quantification of phosphorylated Akt corrected for loading differences (β-tubulin) and normalized to untreated control cells. Data is from one representative donor out of three; and (B-E) show multiplex analysis of cytokine levels produced by peripheral blood monocytes stimulated with 10 ng/ml LPS (B and D, same donor) and 40 μg/ml polyIC (C and E, same donor) after 4 h. (B and C) Stimulated-monocytes were treated with 4 μM ATX-101, 10 μM SB203580 (p38 MAPK inhibitor), and combination of ATX-101 and SB203580. (D and E) Stimulated-monocytes were treated with 4 μM ATX-101, 5 and 10 μM LY294002 (PI3K class I inhibitor), and combination of ATX-101 and LY294002. Measured cytokine levels were normalized to cytokine levels from monocytes stimulated with TLR ligand alone. A two fold or higher increase in cytokine level after TLR stimulation was defined as cytokine induction. # Value is too low to be visible in the column diagram;

cytokine measurement was out of range. Data is from representative experiments out of two.

FIG. 4 shows that ATX-101 reduces Akt phosphorylation and PACT protein levels in HaCaT cells, wherein: (A) shows quantification of Akt Ser473 phosphorylation and PACT protein levels in HaCaT cells treated with 12 μM ATX-101, 2 μg/ml polyIC, and combination of ATX-101 and polyIC for 4 h by Western blot analysis. Protein levels were corrected for loading differences (β-tubulin) and normalized to untreated control cells. Data is from three independent experiments (mean±SD); and (B) shows a confocal fluorescence image of immunofluorescently stained PCNA in HaCaT cells. Bar, 5 μm.

FIG. 5 shows that ATX-101 reduces cytokine secretion of monocytes after TLR stimulation, wherein: (A) and (B) show multiplex analysis of cytokine levels produced by peripheral blood monocytes after 4 h treatment with 4 μM ATX-101 in combination with 10 ng/ml PAM3Cys (A) and 10 μg/ml R848 stimulation (B). Measured cytokine levels were normalized to cytokine levels from monocytes stimulated with TLR ligand alone. A two fold or higher increase in cytokine level after TLR stimulation was defined as cytokine induction.

Cytokine measurement was out of range. Data is from one experiment out of two.

FIG. 6 shows that ATX-A (SEQ ID NO: 1206, which contains the motif RALVK (SEQ ID NO: 1207), which is not capable of interacting with PCNA) reduces less efficiently cytokine secretion of monocytes after TLR stimulation, wherein multiplex analysis of LPS- and polyIC-induced cytokine levels produced by peripheral blood monocytes after 4 h treatment with 4 μM ATX-101 or 4 μM ATX-A in combination with 10 ng/ml LPS and 40 μg/ml polyIC (left and right panel, respectively). Measured cytokine levels were normalized to cytokine levels from monocytes stimulated with TLR ligand alone. Data is from one donor. We obtained similar results for IL-6 secretion in two other donors.

FIG. 7 shows that the Akt inhibitor SC66 reduces cytokine secretion of monocytes to or below the basal levels after TLR stimulation, wherein multiplex analysis of cytokine levels produced by peripheral blood monocytes after 4 h treatment with 4 μM ATX-101 and 1 μg/ml SC66 in combination with 10 ng/ml LPS (left panel) and 40 μg/ml polyIC stimulation (right panel). Measured cytokine levels were normalized to cytokine levels from monocytes stimulated with TLR ligand alone. A two fold or higher increase in cytokine level after TLR stimulation was defined as cytokine induction. # Value is too low to be visible in the column diagram;

Cytokine measurement was out of range. Data is from one experiment out of two.

FIG. 8 shows a graph showing the results of FRET analysis. Normalised FRET (N_(FRET)) measurements are shown between EYFP (yellow fluorescent protein)/ECFP (cyan fluorescent protein) (Lane 1, background due to dimerisation of the tags). EYFP-APIM motif/ECFP-PCNA for various motifs are shown in the other lanes.

EXAMPLES Background to the Experiments

The inventors have determined that more than 20 different signalling kinases, mainly cytoplasmic, contain an APIM sequence, suggesting that they interact with PCNA. These include three direct members of the PI3K/Akt pathway (p110-α, p110-γ, and PI3K-C2β) and several kinases directly or indirectly affecting the MAPK pathways (ERK8, MK2, MK5, MST4, and TAO2). PCNA has heretofore not been linked to signal transduction. The inventors have determined that PCNA plays a role in signal transduction pathways downstream of TLRs. The inventors have established that the signal transduction pathways can be disrupted by targeting PCNA oligopeptidic compounds comprising an APIM motif. These compounds are thought to compete with PCNA for PCNA interacting proteins. The effects of oligopeptidic compounds comprising an APIM motif on signal transduction pathways have been established using an exemplary cell-penetrating APIM-containing peptide ATX-101 (SEQ ID NO: 1198, which contains the APIM motif, RWLVK (SEQ ID NO: 28)).

The inventors have surprisingly found that ATX-101 treatment reduced cytokine secretion of peripheral blood monocytes after stimulation with different TLR ligands and reduced Akt phosphorylation. This data indicates that targeting PCNA affects signal transduction pathways, including the PI3K/Akt and MAPK pathways, likely by inhibition of PCNA-binding of signaling proteins containing the APIM-sequence. These data suggest a regulatory role of PCNA in signal transduction and APIM containing peptides will be useful in the treatment or prevention of a disorder or condition associated with cytokine release from non-proliferating immune cells in blood, such as a disorder or condition resulting in or from, or associated with, hypercytokinemia.

Materials and Methods

Cell Lines

The multiple myeloma cell lines JJN-3 (gift from J. Ball, University of Birmingham, United Kingdom) were grown in RPMI 1640 (Sigma-Aldrich, Schnelldorf, Germany) supplemented with 10% FCS, 2 mM glutamine (Sigma-Aldrich), 2.5 μg/ml amphotericin B (Sigma-Aldrich) and 100 μg/ml gentamicin (Invitrogen, Carlsbad, Calif., USA). HaCaT cells (spontaneously transformed keratinocytes) were cultured in DMEM (Sigma-Aldrich) containing FCS, glutamine, amphotericin B, and gentamicin. All cells were cultured at 37° C. in a humidified atmosphere of 5% CO₂.

Isolation and Stimulation of Peripheral Blood Monocytes

Peripheral blood mononuclear cells were isolated from A+ buffy coats (Blood Bank, St. Olav's University Hospital, Trondheim, Norway) by density gradient centrifugation (Lymphoprep; Axis-Shield PoC, Oslo, Norway). Mononucleated cells were seeded at 4 million cells/ml in serum-free RPMI 1640 supplemented with glutamine and gentamicin. After 60-90 min the adherent cell population was washed before cultured in medium with 10% heat-inactivated human serum (Blood Bank, St. Olav's University Hospital). ATX-101 (4 μM [Innovagen, Lund, Sweden]), ATX-A (4 μM, [Innovagen]) (ATX-A is represented in SEQ ID NO: 1206 and contains the motif RALVK (SEQ ID NO: 1207), which is not capable of interacting with PCNA), p38 inhibitor (SB203580, 10 μM, [Sigma-Aldrich]), PI3K inhibitor (LY294002, 5 and 10 μM, [Sigma-Aldrich]), and Akt inhibitor (Akt inhibitor XVIII SC66, 1 μg/ml, [Sigma-Aldrich]) were added in serum-free medium and incubated for 5 min before LPS (10 ng/ml, [Sigma-Aldrich]), polyIC (40 μg/ml, [GE Healthcare, Little Chalfont, UK]), PAM3Cys (10 ng/ml, [Invivogen, San Diego, Calif., USA]) or R848 (10 μg/ml, [Invivogen]) were added in serum-containing medium. The cells were incubated for 4 h before the supernatants were harvested and frozen prior to further cytokine analysis by the 27-plex assay (Bio-Plex Pro™ Human cytokine 27-plex assay).

Preparation of Cell Extracts and Western Analysis

Mononucleated cells were seeded and stimulated as described above. JJN-3 cells were treated with ATX-101 (6 μM) for 4 h. HaCaT cells were treated with ATX-101 (12 μM), polyIC (2 μg/ml), and LPS (100 ng/ml) for 4 h. The cells were harvested, the cell pellet was resuspended in 1× packed cell volume of buffer 1 (10 mM Tris-HCl pH 8.0, 200 mM KCl) and diluted in the same volume (packed cell volume+buffer 1) of buffer 2 (10 mM Tris-HCl pH 8.0, 200 mM KCl, 10 mM EGTA, 10 mM MgCl₂, 40% glycerol, 0.5% NP40, 1 mM DTT, 1% phosphatase inhibitor cocktails 1 and 3 [Sigma-Aldrich], 2% Complete EDTA-free protease inhibitor [Roche, Oslo, Norway], and 2 μl/ml Omnicleave [Epicentre Technologies, Madison, Wis., USA]). After incubation for 1.5 h at 4° C., the cell extracts were centrifuged at 14,000 rpm for 10 min. Supernatants were collected and separated on 10% Bis-Tris-HCl gels (NuPAGE, Invitrogen). After gel electrophoresis, the polyvinylidene fluoride membranes (Immobilion, Millipore, Oslo, Norway) were blocked in 50% Odyssey blocking buffer (LI-COR Bioscience, Cambridge, UK) in TBS. The primary antibodies against (a) P-Akt (monoclonal, Cell Signaling, Beverly, Mass., USA), PACT (monoclonal, Santa Cruz biotechnology Inc., Dallas, Tex., USA), P-p70 S6 Kinase (monoclonal, Cell Signaling), P-ERK1/2 (monoclonal, Cell Signaling), and β-tubulin (polyclonal, Abcam, Cambridge, UK), as well as the fluorescently labeled secondary antibodies, goat α-rabbit 680RD and goat α-mouse 800CW (LI-COR) were diluted in 20% Odyssey blocking buffer in TBST (TBS with 0.1% Tween 20). The proteins were visualized with the Odyssey infrared imaging system (LI-COR) and quantified using Odyssey Image Studio V2. Protein levels were compared to the protein level in untreated cells, which was set to 100%. β-tubulin was used as reference for data normalization.

Immunofluorescence and Confocal Imaging

HaCaT cells and peripheral blood monocytes were grown on glass bottom dishes and stained as described using an antibody α-PCNA (PC10, Santa Cruz biotechnology Inc.; 1:1000) and Alexa fluor 532 goat α-mouse (Invitrogen). The nuclei were stained with DRAQ5 prior to imaging according to the manufacturer's manual (eBioscience, San Diego, Calif., USA). The fluorescent images were acquired using a Zeiss LSM 510 Meta laser scanning microscope equipped with a Plan-Apochromate 63×/1.4 oil immersion objective in 2% FCS in PBS at RT using the Zeiss LSM 510 software. The stained cells were excited at λ=543 nm and detected at λ>=560-615 for Alexa fluor 532. DRAQ5 was excited at λ=633 nm and detected at λ>650 nm. The thickness of the slice was 1 μm. All images were acquired with consecutive scans. No image processing was performed, except contrast and intensity adjustments.

IFN-β ELISA

Monocytes were stimulated as described above. IFN-β was measured in supernatants with the VeriKine-HSTMHuman IFN-β Serum ELISA kit (Pestka Biomedical Laboratories, Piscataway, N.J., USA) according to the manufacturer's instructions with the following adaptions: the amount of supernatant was doubled from 50 to 100 μl; sample buffer was reduced from 50 to 25 μl; and antibody solution was reduced from 50 to 25 μl (reducing assay diluent, keeping same volume of antibody concentrate and diluent additive). The OD was measured at 450 nm.

Cell Survival Assay

Cells were seeded into 96-well plates and ATX-101, kinase inhibitors (SB203580 and BIRB0796), and polyIC were added alone or in combination at the indicated concentrations. Cells were exposed continuously and harvested every day for the next four days using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2.5 diphenyl-tetrazolium bromide) assay (Gilljam et al., 2009, supra). The average from at least 4 wells was used to calculate cell survival.

Example 1: Targeting PCNA with APIM-Containing Peptides Affects Signal Transduction in Multiple Myeloma Cells

Multiple myeloma cells secrete cytokines and respond to various growth-stimulatory factors from their tumor microenvironment in positive feedback loops that support myeloma cell growth. The MAPK and PI3K/Akt pathways have been suggested as potential therapeutic targets in multiple myeloma, alone and in combination because these pathways are frequently dysregulated and extensive cross-talk between these pathways exists. Furthermore, these pathways are important for cytokine production.

We found that ATX-101 increased the growth-inhibitory effect of p38 MAPK and class I PI3K inhibitors in the multiple myeloma cell line JJN-3 (FIG. 1A). In addition, ATX-101 increased the growth-inhibitory effects of ERK5 and MAPK kinase 1 (MEK1) inhibitors, and enhanced the cytotoxic effects of melphalan in combination with these inhibitors as well as the p38 inhibitor in another multiple myeloma cell line (RPMI-8226). To further elucidate whether ATX-101 directly affected the MAPK and PI3K pathways, we analyzed the phosphorylation levels of downstream kinases in JJN-3 cells at different time points after ATX-101 addition. The phosphorylation of Akt and the p70 S6 kinase (downstream of mTOR) were both decreased by ATX-101 treatment. In contrast, a transient increase in ERK1/2 phosphorylation was detected upon ATX-101 treatment (FIG. 1B).

These results indicate that ATX-101 affects important signal transduction pathways in multiple myeloma cells including the PI3K/Akt/mTOR and MAPK pathways.

Example 2: Targeting PCNA with APIM-Containing Peptides Reduces Cytokine Secretion of Monocytes after TLR Stimulation

Next, we investigated whether ATX-101 could affect cytokine secretion from primary monocytes, which is partly regulated by the PI3K/Akt and MAPK pathways. We stimulated peripheral blood monocytes with different TLR ligands to induce cytokine production and measured the concentration of 27 cytokines in the cell culture supernatants. The secretion of LPS-induced cytokines was efficiently reduced by ATX-101 addition (FIG. 2A). Similarly, ATX-101 treatment reduced the secretion of CXCL10, CCL2, CCL3, and CCL4 after TLR3 stimulation with polyIC (FIG. 2B). We found similar reduction in cytokine secretion induced by the TLR7/8 ligand R848 and the TLR2 ligand PAM3Cys (FIG. 5). These results show that targeting PCNA with APIM-containing peptides affects cytokine production. The mechanism is likely inhibition of multiple kinase-PCNA interactions, affecting signaling pathways downstream of the TLR adaptor proteins because ATX-101 treatment reduced cytokine secretion downstream of TLRs dependent on both MyD88 and TRIF.

ATX-101 treatment decreased the CXCL10 secretion induced by polyIC and also the basal level production of this cytokine from isolated monocytes (FIGS. 2A and B). We measured the IFN-β levels in supernatants from LPS- and polyIC-stimulated monocytes because the induction of CXCL10 expression is a secondary event and requires the initial secretion of IFN-β. Consistent with the reduced CXCL10 secretion, ATX-101 treatment also decreased the IFN-β secretion, supporting that ATX-101 affects signaling downstream of the TLR adaptor TRIF (FIG. 2C). Importantly, ATX-101 treatment at the used concentration did not induce apoptosis in monocytes. Furthermore, a mutant version of ATX-101, ATX-A that does not bind to PCNA, was unable or less efficient in decreasing the secretion of cytokines compared to ATX-101 (FIG. 6). These results therefore indicate that ATX-101 affects cytokine secretion by specifically targeting PCNA. Monocytes contain substantial amounts of PCNA in the cytoplasm as compared to for example HeLa cells (FIG. 2D). Collectively, these data show that PCNA may have a regulatory role in the innate immune response.

Example 3: Reduction of Akt Phosphorylation by APIM-Containing Peptides is Likely Affecting the Cytokine Production in TLR-Stimulated Monocytes

Activation of NF-κB, MAPKs, and the PI3K/Akt pathway are important features after TLR activation, and our results indicate that ATX-101 affects two of these pathways in the multiple myeloma cell line JJN-3 (FIG. 1). To examine whether ATX-101 affected the PI3K/Akt pathway in monocytes similar to JJN-3 cells, we analyzed Akt phosphorylation levels in monocytes. LPS- and polyIC-induced Akt phosphorylation as well as the basal Akt phosphorylation level was reduced by ATX-101 treatment (FIG. 3A). To further investigate whether ATX-101 reduced or increased the effect of specific inhibitors of PI3K, Akt, and p38 MAPK with regard to cytokine production similar to the reduced cell growth observed in JJN-3 cells (FIG. 1A), we measured cytokine secretion from LPS- and polyIC-stimulated monocytes in presence of kinase inhibitors alone and in combination with ATX-101.

ATX-101 and a p38 MAPK inhibitor both reduced cytokine secretion from stimulated monocytes, and the combination of ATX-101 and inhibitor resulted in an increased reduction of most cytokines (FIGS. 3B and C). The PI3K inhibitor LY294002 on the other hand had less effect on induced cytokine secretion alone and in combination with ATX-101, but still the combination showed a reduction in the secretion of some cytokines (e.g. IFNγ, IL-1RA, IL-2, 4, and 9 after LPS, and IL-1RA, CCL2, 3, and 4 after polyIC stimulation) (FIGS. 3D and E). Direct inhibition of Akt, the central kinase further downstream in the PI3K pathway reduced the secretion of all cytokines to or below the basal levels, indicating a vital role in cytokine regulation (FIG. 7); however, this strong single agent response of the inhibitor (in absence of apoptosis) did not allow examination of its combination with ATX-101. In summary, these results verify that both the p38 MAPK and PI3K/Akt pathway are important for cytokine secretion from monocytes, and indicate that targeting PCNA with APIM-peptides enhances the effect of inhibiting these pathways. Thus, the results shown in FIG. 1, i.e. increased inhibition of cell growth by ATX-101 alone and in combination with MAPK and PI3K pathway inhibitors and reduced Akt activation after ATX-101 treatment in JJN-3 cells, correlate with reduced cytokine production and reduced Akt activation in monocytes. These data further support that PCNA has a regulatory role in signal transduction, affecting both cell growth and cytokine production.

Example 4: APIM-Containing Peptides Reduce Akt Phosphorylation and PACT Protein Levels in HaCaT Keratinocytes

To further support the possible regulatory role of PCNA in the cellular response to TLR agonists, we used the human keratinocyte cell line HaCaT that expresses functional TLR3 and is responsive to polyIC treatment. We examined Akt phosphorylation levels in HaCaT cells and found that ATX-101 treatment reduced Akt phosphorylation in unstimulated and stimulated cells similar to JJN-3 cells and monocytes (FIG. 4A). Human keratinocytes respond to polyIC via the known dsRNA sensing receptors TLR3, PKR, RIG-I, and MDA5. The dsRNA-binding APIM-containing protein PACT is an activator of PKR and RIG-I. Thus, PACT may interact with PCNA and this interaction may have a regulatory role in the response to polyIC in HaCaT cells. We therefore examined PACT protein levels in HaCaT cells treated with polyIC and ATX-101, and a combination of both.

ATX-101 addition to unstimulated cells had no clear effect on the PACT protein level, but the addition of ATX-101 further reduced the protein level of PACT in polyIC-stimulated cells (FIG. 4A). The mutant peptide ATX-A had less effect on Akt phosphorylation and PACT protein levels than ATX-101 similarly to what we observed for cytokine secretion. Thus, these data indicate that APIM-peptides affect the PI3K/Akt pathway as well as PACT protein levels in response to polyIC in HaCaT cells. We hypothesize that APIM-peptides interfere with several signal transduction pathways by targeting PCNA's interactions with cytoplasmic signaling proteins such as PACT. PCNA is mainly detected in the nucleus of HaCaT cells, but some cells also contain PCNA in the cytoplasm (FIG. 4B).

Discussion

We show that targeting PCNA with an APIM-containing peptide, e.g. ATX-101, affects the cellular response to TLR agonists, supporting a role for PCNA in cellular stress responses. ATX-101 treatment reduced the cytokine secretion from monocytes and decreased Akt phosphorylation in monocytes and HaCaT cells stimulated with different TLR ligands. The APIM-sequence is found in over 200 proteins including several signaling kinases that have been described to act in TLR signaling. Therefore, ATX-101 likely affects several signal transduction pathways.

Our data indicate that ATX-101 down regulates the PI3K/Akt pathway, a known regulator of cytokine production as shown by using the Akt inhibitor SC66. Thus, the reduced Akt activation likely contributes to the decreased cytokine secretion from TLR-stimulated monocytes upon ATX-101 treatment.

In our experiments, we observed no strong induction of IL-12 by the TLR ligands tested; however, ATX-101 treatment reduced the basal level of IL-12 (FIG. 2A and FIG. 5). Thus, ATX-101 may also affect cytokine secretion by interfering with PI signaling.

Our data suggest that ATX-101 did not reduce cytokine secretion by its cell-penetrating peptide but rather by targeting PCNA via the APIM-sequence because the mutant ATX-A with a single amino acid change in APIM reduced cytokine secretion far less than ATX-101. The small observed effects of ATX-A are likely because ATX-A still has some weak PCNA-binding ability.

We initially found that ATX-101 interfered with signal transduction pathways including the PI3K/Akt pathway in the multiple myeloma cell line JJN-3 independent of TLR stimulation. The ATX-101-mediated effect on signal transduction pathways is therefore not restricted to TLR signaling. The cellular target of ATX-101 is PCNA and thus our data suggest a new regulatory function of PCNA in signaling pathways likely by acting as cytoplasmic binding platform. Thus, APIM-mediated PCNA interactions play a role in the cellular stress response to pathogens and damage-associated molecular patterns recognized by TLRs. ATX-101 reduces cytokine secretion from monocytes at doses that do not induce apoptosis.

Example 5: In Silico Characterisation of APIM Consensus Motif

The inventors have performed sequence analyses to determine how much variation within the APIM motif occurs naturally, i.e. in native sequences across a number of species. As PCNA is highly conserved across eukaryotic organisms, it is expected that sequence variation of the APIM motif in orthologues of polypeptides that are thought to interact with PCNA is representative of the variation that may be used in the oligopeptidic compounds of the invention, i.e. variation of amino acids within the APIM motif at some positions, particularly X₃ and X₄, may be permitted without losing affinity to PCNA.

The inventors used identified 657 human polypeptide sequences that comprise the motif [K/R]-[F/W/Y]-[A/L/V/I]-[A/L/V/I]-[K/R] (SEQ ID NO:19) from a possible 21,673 polypeptide sequences. Of the 657 sequences identified, 291 were excluded because insignificant information about the function of the polypeptides was available. The remaining 366 were considered to be polypeptides that are likely to interact with PCNA and these sequences were used to identify orthologues in: Bos taurus (288 orthologues); Rattus norvegicus (286 orthologues); Mus musculus (312 orthologues); Gallus gallus (236 orthologues); Xenopus tropicalis (200 orthologues); Danio rerio (189 orthologues); Caenrhabditis elegans (102 orthologues); Drosophila melanogaster (136 orthologues); and Saccharomyces cerevisiae (65 orthologues). Alignment of the domains of the orthologues that comprise the APIM motif suggested that the motif may defined as:

[R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T/N/Q/C]-[L/I/V/A/M/G/S/T/N/Q/R/H/K/C]-[K/R/H/P] (SEQ ID NO: 2),

wherein specific combinations of amino acids at positions 3 and 4 that were identified in the orthologues include:

LL, LA, LV, AL, VL, VI, LI, IL, VV, VA, IV, II, AV, IA, AI, AM, LM, LS, LT, IS, MV, TV, AA, IM, LN, LQ, VM, TL, SL, IT, VT, LG, MA, ML, NL, QL, QI, TI, SI, AS, VS, SV, CA, IG, LR, VR, TK and IR. Particularly common combinations are LL, LA, LV, AL, VL, VI, LI, IL, VV, VA, IV, II, AV, IA, AI, AM, LM, LS and LT, the most common being LL, LA, LV, AL, VL, VI, LI, IL, VV, VA, IV, II, AV, IA and AI.

Thus, the broadest definition of the APIM motif was derived from this analysis, and all polypeptides comprising an APIM motif according to this definition could reasonably be expected to interact with, i.e. bind to, PCNA.

Example 6: In Vivo Characterisation of APIM Consensus Motif

This work described in this Example investigates interaction between APIM peptides and PCNA.

In living S-phase cells, PCNA tagged with green fluorescent protein (EGFP) forms distinct foci representing sites of replication and thus can be used as a S-phase marker.

PCNA tagged with cyan fluorescent protein (ECFP) was co-expressed with various APIM peptide constructs fused with yellow fluorescent protein (EYFP). To examine the degree of proximity of APIM peptides and PCNA, fluorescence resonance energy transfer (FRET) was measured.

Live HeLa cells were examined 16-24 hours after transient transfection (by Fugene 6 (Roche Inc.) according to the manufacturer's recommendations) of ECFP and EYFP fusion constructs. Fluorescent images were acquired using a Zeiss LSM 510 Meta laser scanning microscope equipped with a Plan-Apochromate 63×/1.4 oil immersion objective. Enhanced cyan fluorescent protein (ECFP) was excited at λ=458 nm and detected at λ=470-500 nm and enhanced yellow fluorescent protein (EYFP) was excited at λ=514 nm and detected at λ=530-600 nm, using consecutive scans. The thickness of the slice was 1 μm.

Fluorescent resonance energy transfer (FRET) occurs if the tags (EYFP and ECFP) are less than 100 Å (10 nm) apart. We detected FRET using the sensitised emission method, measuring acceptor (EYFP) emission upon donor (ECFP) excitation. We had FRET when the intensity of emitted light from EYFP after excitation of the ECFP fluorochrome was stronger than the light emitted by ECFP or EYFP-tagged proteins alone, after excitation with the EYFP and ECFP lasers respectively (bleed through), given by the equation: FRET=I₂−I₁(I_(D2)/I_(D1))−I₃(I_(A2)/I_(A3)) is >0. FRET was normalised for expression levels using the equation: N_(FRET)=FRET/(I₁×I₃)^(1/2). N_(FRET) was calculated from mean intensities (I) within a region of interest (ROI) containing more than 25 pixels where all pixels had intensities below 250 and the average intensities were between 100 and 200 for both the donor and the acceptor constructs. Channel 1 (ECFP) and 3 (EYFP) were measured as described above for imaging, and channel 2 (FRET) was excited with λ=458 nm and detected at λ=530-600 nm. I_(D1, D2, D3) and I_(A1, A2, A3) were determined for cells transfected with ECFP and EYFP constructs only, with same settings and same fluorescence intensities as co-transfected cells (I₁ and I₃). ECFP-PCNA and EYFP-PCNA were included as positive controls, and due to dimerisation of co-expressed tags, ECFP and EYFP proteins expressed from empty vectors were included as negative controls in all experiments.

FIG. 8 shows that a significant FRET signal could be detected for all of the variants tested, which verifies that a variety of peptides within the APIM motif definition described herein (and that occur in polypeptides that are expected to interact with PCNA) are capable of interacting with PCNA and would therefore be expected to find utility in the method and uses described herein, i.e. as immunosuppressive peptides, e.g. for inhibiting or preventing cytokine release for non-proliferating immune cells in blood and treating or preventing disorders or conditions associated therewith.

Example 7: Identification of Cytosolic Proteins Involved in Cellular Signalling that Interact with PCNA

Several cytosolic proteins contain APIM suggesting that they may interact with PCNA. In order to investigate the PCNA-“interactome” further, the inventor performed PCNA co-immunoprecipitation (co-IP) experiments on extracts from HaCaT cells crosslinked using a low concentration of formaldehyde to preserve protein complexes in their in vivo conformation. The eluate was separated on a gel, and the gel pieces were subjected to tryptic digestion and Orbitrap-MS. A mock antibody specific for TOM20 was used in a control co-IP assay. Relevant proteins detected by MS in the PCNA pull down, but not in the mock pull down, are listed in Table 3.

APIM containing proteins detected include MST4 and PLK3, which are involved in regulating ERK and Akt respectively. PACT protein was also detected. These proteins are involved in cytokine and chemokine expression pathways. PCNA also pulled down the APIM containing protein BRE, a protein part of the BRCA-1-complex in the nuclei and the BRISK-complex in the cytosol, which were recently found to be involved in regulation of TNF-induced apoptosis. One interesting PIP-box protein identified was the TAB2 which is involved in positive feedback in a kinase complex in NFkB signalling. Overall, 20 APIM-containing proteins and 23 PIP-box containing proteins were detected exclusively in the PCNA pull down in absence of cellular stress.

A mock antibody should ideally target an unrelated protein. However, as PCNA has many interaction partners and likely acts as a signalling hub in the cytoplasm, an ideal mock antibody is difficult to obtain. Indeed, the mock protein chosen here (TOM20) was detected in the PCNA pull down by MS (Mascot-score 32.53, not detected by Sequest; data not shown). Table 4 includes several interesting proteins that are not exclusively found, but enriched, in the PCNA pull down compared to mock pull down, based on the number of detected peptides and Mascot/Sequest score. These include several proteins in the MAPK pathways, including TAO3, MAPKK3, ERK1/2 and the APIM-containing proteins MK2 and TAO1. Akt1 and p38 alpha were also enriched in PCNA pull down, however, these proteins were detected in gel pieces corresponding to different molecular weights from the α-PCNA and α-mock pull down suggesting that regulation by PTMs is important for their localization to PCNA complexes. The APIM-protein PACT was detected from the gel-piece with the expected molecular weight (MW) of PACT in the PCNA pull down only (Table 3). PACT was found in both PCNA and mock pull down (less in mock), and in a gel piece with 5-10 kDa higher molecular weight than predicted (Table 4). The increase in molecule weight suggests that PACT may be regulated by PTMs, e.g. polyubiquitination which leads to degradation. Stimulation of HaCat cells with both polyIC and ATX-101, and the combination, led to rapid (4 h) degradation of PACT (FIG. 4A). Overall, the data suggest that PCNA and PACT are in a common complex, directly interacting via APIM. A full list of proteins containing an APIM or PIP-box motif identified in the pull-down assay is set out in Table 5.

TABLE 3 (Unique peptides disclosed as SEQ ID NOS 1221-1230, respectively, in order of appearance) Proteins only in PCNA pull down Unique peptides Fraction Predicted Mw APIM-proteins PACT AAEAAINILK 30-40 kDa 34.4 kDa BRE VGLDATNCLR 40-45 kDa 43.6 kDa SGCTSLTPGPNCDR NNWTGEFSAR ELVQQYHQFQCSR MST4 SIAVAEAACPGITDK 45-55 kDa 46.5 kDa TQQVVAIK NQAIEELEK PLK3 CIKQVHYTLPASLSLPARQLLAAILR >150 kDa 71.6 kDa PIP-proteins TAB2 TSSTSSSVNSQTLNR 65-90 kDa 76.5 kDa

TABLE 4 (Unique peptides disclosed as SEQ ID NOS 1221 and 1231-1235, respectively,  in order of appearance) Proteins enriched in PCNA pull down APIM- Predicted Score PCNA Score Tom20 proteins Unique peptides Fraction Mw (Mascot/Sequest (Mascot/Sequest) PACT AAEAAINILK 40-45 kDa  34.4 kDa 23.03/2.06 25.12/1.61 LPEYTLSQEGGPAHK** MK2 IEDASN PLLLK* 40-45 kDa  45.6 kDa 27.56/6.33 -/1.79 TAO1 LDEAQEAECQVLK** 65-90 kDa 116.0 kDa 49.83/11.10 -/2.09 EIEAFDSESMR** KFQQHIQAQQK***

TABLE 5 Proteins containing APIM or PIP in pull down using anti-PCNA* from unstimulated, weakly crosslinked HaCat cells: APIM-proteins PIP-box proteins Uniprot ID Name Uniprot ID Name B2RTY4 Unconventional myosin-IXa O15541 RING finger protein 113A O60573 Eukaryotic translation initiation factor 4E type 2 O43502 DNA repair protein RAD51 homolog 3 O94887 FERM, RhoGEF and pleckstrin domain-containing O43929 Origin recognition complex subunit 4 protein 2 P18858 DNA ligase 1 O60256 Phosphoribosyl pyrophosphate synthase-associated protein 2 P55196 Afadin O60303 Uncharacterized protein KIAA0556 Q09327 Beta-1,4-mannosyl-glycoprotein 4-beta- O60890 Oligophrenin-1 N-acetylglucosaminyltransferase Q15051 IQ calmodulin-binding motif-containing protein 1 P18858 DNA ligase 1 Q4AC94 C2 domain-containing protein 3 P19474 E3 ubiquitin-protein ligase TRIM21 Q5TBA9 Protein furry homolog P20585 DNA mismatch repair protein Msh3 Q8IX01 SURP and G-patch domain-containing protein 2 P24386 Rab proteins geranylgeranyltransferase component A1 Q8NB46 Serine/threonine-protein phosphatase 6 regulatory Q14558 Phosphoribosyl pyrophosphate synthase-associated protein 1 ankyrin repeat subunit C Q9H4B4 Serine/threonine-protein kinase PLK3 Q15172 Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit alpha isoform Q9HCE6 Rho guanine nucleotide exchange factor 10-like protein Q1ED39 Lysine-rich nucleolar protein 1 Q9NQW1 Protein transport protein Sec31B Q4KMP7 TBC1 domain family member 10B Q9NXR7 BRCA1-A complex subunit BRE Q6VN20 Ran-binding protein 10 Q9NYT0 Pleckstrin-2 Q96DF8 Protein DGCR14 Q9P289 Serine/threonine-protein kinase MST4 Q96S59 Ran-binding protein 9 Q9UKN7 Unconventional myosin-XV Q9H0U9 Testis-specific Y-encoded-like protein 1 Q9UL63 Muskelin Q9HBU6 Ethanolamine kinase 1 Q9Y6J0 Calcineurin-binding protein cabin-1 Q9NYJ8 TGF-beta-activated kinase 1 and MAP3K7-binding protein 2 Q9UIF7 A/G-specific adenine DNA glycosylase Q9UKN7 Unconventional myosin-XV Q9UNZ2 NSFL1 cofactor p47 *These proteins are not detected in MOCK (anti-TOM20) IP

Example 8: Determination of the Level of Apoptosis in Non-Proliferating Immune Cells (Monocytes) from Blood when Treated with APIM-Containing Peptides

Annexin staining was used to determine cell viability and cells were counted using flow cytometry.

Peripheral blood lymphocytes were isolated from A+ buffy coats (Blood Bank, St. Olav's University Hospital) by density gradient centrifugation (Lymphoprep; Axis-Shield PoC, Oslo, Norway). The adherent cell fraction after 90 minute incubation, termed monocytes, were washed twice with HANKS and maintained in RPMI 1640 supplemented with 2 mM glutamine, 100 mg/ml gentamicin and 25% heat-inactivated HS over-night. Cells were cultured at 37° C. in a humidified atmosphere of 5% CO₂ over-night. The next day cells were treated with different concentrations of APIM-containing peptides ATX-101 (SEQ ID NO: 1198) and ATX-P (MDRWLVPWKKKRKIRRRRRRRRRRR, SEQ ID NO: 1208). An R₁₁ peptide and no peptide treatment were used as controls. After 4 hours, the cells were washed once in PBS, and re-suspended in buffer as recommended by the manufacturer of the Annexin V-Pacific Blue kit (Invitrogen). Cells were identified monocytes according to size (FSC) and granularity (SSC).

Table 6 shows that there was no significant increase in annexin positive cells (apoptotic cells) when treated with APIM peptides.

Furthermore, as discussed herein, a 4 μM dose of ATX-101 reduces the cytokine production from TLS-ligand stimulated monocytes in absence of apoptosis. It is noted that the same dose showed low toxicity on normal cells in BMCS assays (Müller et al, PloS One, 2013, v. 8: e70430).

TABLE 6 Percentage of annexin positive cells Control (no peptide) 67% Peptide 5 μM peptide treatment 10 μM peptide treatment R11 (negative control) 55% 56% ATX-101 (SEQ ID NO: 1198) 72% 79% ATX-P (SEQ ID NO: 1208) 65% 81%

Example 9: Determination of APIM Peptide Interaction with PCNA

Microscale thermophoresis (MST) was used to determine the dissociation constant for various APIM containing peptides.

PCNA was labeled with a fluorescent molecule. The concentration of PCNA was kept constant, whereas dilutions of each APIM containing peptide were prepared (1:1). In a mix of protein and peptide, the signal was recorded in all capillaries with varying concentrations of the unlabeled peptide, and any change of thermophoretic properties was observed as a change in fluorescence intensity.

Table 7 shows that ATX-101 and ATX-101-P (natural variant of APIM in ABH2) both show stronger interactions with PCNA than ATX-A, a negative control (a low Kd value indicates a strong interaction, whereas a high Kd value indicates a weak interaction). Furthermore, no data on specific interaction with R11 could be obtained by MST, indicating that this peptide does not interact with PCNA.

TABLE 7 Peptide Kd ATX-101 177 ATX-101-P 123 ATX-A 769 R11 nd 

The invention claimed is:
 1. A method of treating or preventing hypercytokinemia resulting from cytokine release from non-proliferating immune cells in blood, said method comprising administering an agent, or a composition containing an agent, to a subject in need thereof, wherein said agent comprises: (i) an oligopeptidic compound comprising a PCNA interacting motif and a domain that facilitates the cellular uptake of said compound, wherein the PCNA interacting motif is X₁X₂X₃X₄X₅ (SEQ ID NO: 1) and wherein: X₁ is a basic amino acid; X₂ is an aromatic amino acid; X₃ is an uncharged amino acid other than an aromatic amino acid, Glycine (G) or Proline (P); X₄ is any amino acid other than Proline (P), Glutamine (Q), an acidic amino acid or an aromatic amino acid; and X₅ is a basic amino acid or Proline (P), and wherein said PCNA interacting motif is not KYILK (SEQ ID NO: 234); or (ii) a nucleic acid molecule comprising a sequence encoding the oligopeptidic compound of (i), wherein said subject has an infection or infectious disease and wherein the infection or infectious disease is not a viral-induced hyperproliferative disease.
 2. The method of claim 1, wherein said agent or composition is provided as a combined preparation with one or more additional active agents for separate, simultaneous or sequential use or administration.
 3. The method of claim 1, wherein said non-proliferating immune cells display the cell surface receptor CD14 and/or the cell surface receptor CD16.
 4. The method of claim 1, wherein said non-proliferating immune cells are monocytes.
 5. The method of claim 2, wherein the one or more additional active agents is a kinase inhibitor, an immunosuppressive compound, an anti-inflammatory compound, anti-microbial compound or a steroid.
 6. The method of claim 1, wherein the infection or infectious disease is: (i) a microbial infection or microbial infectious disease, (ii) a viral infection or viral infectious disease other than a viral-induced hyperproliferative disease; or (iii) a parasitic infection or parasitic infectious disease.
 7. The method of claim 6, wherein the microbial infection or microbial infectious disease is a fungal infection or fungal infectious disease.
 8. The method of claim 1, wherein the PCNA interacting motif comprises a sequence as set forth in (SEQ ID NO: 2); wherein the amino acid at position 4 is not glutamine and the PCNA interacting motif is not KYILK (SEQ ID NO: 234).
 9. The method of claim 1, wherein the PCNA interacting motif comprises a sequence selected from any one of SEQ ID NOs: 22 to 233 or 235 to
 297. 10. The method of claim 1, wherein the domain that facilitates the cellular uptake of the oligopeptidic compound is a cell penetrating peptide (CPP).
 11. The method of claim 10, wherein the CPP is selected from any one of SEQ ID NOs: 302 to 1162 or a fragment and/or derivative thereof.
 12. The method of claim 1, wherein the agent further comprises a linker domain.
 13. The method of claim 12, wherein the linker domain comprises a nuclear localisation signal sequence.
 14. The method of claim 1, wherein the agent comprises a PCNA interacting motif as set forth in SEQ ID NO: 28, a linker or nuclear localisation signal sequence as set forth in SEQ ID NO: 1176 and a cell penetrating signal sequence as set forth in SEQ ID NO:
 337. 15. The method of claim 1, wherein the agent comprises a sequence as set forth in any one of SEQ ID NOs: 1182 to
 1204. 16. The method of claim 1, wherein the subject is suffering from sepsis.
 17. The method of claim 6, wherein the viral infection or viral infectious disease is AIDS/HIV, Dengue fever, measles, mumps, rubella, influenza or hepatitis.
 18. The method of claim 6, wherein the parasitic infection or parasitic infectious disease is malaria, toxoplasmosis, trypanosomiasis or schistosomiasis.
 19. The method of claim 1, wherein the agent comprises a sequence as set forth in SEQ ID NO: 1198, 1203 or
 1204. 20. The method of claim 1, wherein the PCNA interacting motif comprises the sequence of SEQ ID NO:
 1209. 21. The method of claim 10, wherein the CPP is selected from any one of SEQ ID NOs: 1210 to 1220 or a fragment and/or derivative thereof.
 22. The method of claim 1, wherein the agent comprises a sequence as set forth in SEQ ID NO:
 1208. 